Fields Virology, 7th ed., Volume 4 - Fundamentals [7 ed.] 9781975112516

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Fields VIROLOGY VOLUME 4: Fundamentals SEVENTH EDITION EDITORS-IN-CHIEF Peter M. Howley, MD Shattuck Professor of Pathological Anatomy Departments of Immunology and Pathology Harvard Medical School Boston, Massachusetts David M. Knipe, PhD Higgins Professor of Microbiology and Molecular Genetics Head, Harvard Program in Virology Department of Microbiology Blavatnik Institute Harvard Medical School Boston, Massachusetts ASSOCIATE VOLUME EDITORS L. W. Enquist, PhD Emeritus Professor Department of Molecular Biology Princeton University Princeton, New Jersey Jeffrey I. Cohen, MD Chief, Laboratory of Infectious Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland Eric O. Freed, PhD HIV Dynamics and Replication Program Center for Cancer Research National Cancer Institute Frederick, Maryland Blossom Damania, PhD Boshamer Distinguished Professor Vice Dean for Research, School of Medicine Department of Microbiology and Immunology University of North Carolina at Chapel Hill Chapel Hill, North Carolina Sean P. J. Whelan, PhD Marvin A. Brennecke Distinguished Professor Chair, Molecular Microbiology School of Medicine Washington University in St. Louis St. Louis, Missouri

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Contributors Jônatas Santos Abrahão, PhD Professor Universidade Federal de Minas Gerais Belo Horizonte, Minas Gerais, Brazil James C. Alwine, BS, PhD Emeritus Professor of Cancer Biology Cancer Biology University of Pennsylvania Philadelphia, Pennsylvania Thomas J. Braciale, MD, PhD Professor Department of Pathology University of Virginia Medical Center Charlottesville, Virginia Dennis R. Burton, BAOxon, PhD Professor and Chair Immunology & Microbiology The Scripps Research Institute La Jolla, California Byron Caughey, PhD Senior Investigator Rocky Mountain Laboratories National Institute of Allergy and Infectious Diseases National Institutes of Health Hamilton, Montana Donald M. Coen, PhD Professor Department of Biological Chemistry and Molecular Pharmacology Blavatnik Institute Harvard Medical School Boston, Massachusetts Richard C. Condit, PhD Emeritus Professor Department of Molecular Genetics & Microbiology University of Florida Gainesville, Florida James E. Crowe Jr, MD Ann Scott Carell Chair Department of Pediatrics, Pathology, Microbiology, and Immunology Director, Vanderbilt Vaccine Center Vanderbilt University Medical Center University Distinguished Professor of Chemistry Vanderbilt University Nashville, Tennessee Daniel DiMaio, MD, PhD Waldemar Von Zedtwitz Professor of Genetics Yale University New Haven, Connecticut L. W. Enquist, PhD Emeritus Professor Department of Molecular Biology Princeton University Princeton, New Jersey

Paul D. Friesen, PhD Professor Department of Biochemistry Institute for Molecular Virology University of Wisconsin–Madison Madison, Wisconsin Alexander L. Greninger, MD, PhD, MPhil, MS Larry Corey Endowed Assistant Professor Department of Laboratory Medicine and Pathology University of Washington Seattle, Washington Young S. Hahn, PhD Professor Department of Microbiology, Immunology, and Cancer Biology University of Virginia School of Medicine Charlottesville, Virginia Stephen C. Harrison, PhD Giovanni Armenise–Harvard Professor in Basic Biomedical Science Harvard Medical School Boston Children’s Hospital Howard Hughes Medical Institute Boston, Massachusetts Graham F. Hatfull, PhD Professor Department of Biological Sciences University of Pittsburgh Pittsburgh, Pennsylvania Mark T. Heise, PhD Professor Departments of Genetics, and Microbiology and Immunology The University of North Carolina at Chapel Hill Chapel Hill, North Carolina Eric Hunter, PhD Professor Pathology and Laboratory Medicine Emory University Atlanta, Georgia Sun Hur, PhD Professor Howard Hughes Medical Institute Harvard Medical School Boston, Massachusetts Akiko Iwasaki, PhD Sterling Professor of Immunobiology Yale University School of Medicine Investigator, Howard Hughes Medical Institute Department of Immunobiology Yale University New Haven, Connecticut Keith R. Jerome, MD, PhD Larry Corey Endowed Professor in Virology Vaccine and Infectious Disease Division Fred Hutchinson Cancer Center Head Virology Division Department of Laboratory Medicine and Pathology University of Washington Seattle, Washington

Eugene V. Koonin, PhD NIH Distinguished Investigator National Center for Biotechnology Information National Institutes of Health Bethesda, Maryland Mart Krupovic, PhD Associate Professor Archaeal Virology Unit Institut Pasteur Paris, France Daniel R. Kuritzkes, MD Chief, Division of Infectious Diseases Harriet Ryan Albee Professor of Medicine Division of Infectious Diseases Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts Yi Li, PhD Professor School of Life Sciences Peking University Beijing, China Kristiina Mäkinen Professor of Plant Pathology Department of Microbiology University of Helsinki Helsinki, Finland Jason Mercer, PhD Professor of Virus Cell Biology Institute of Microbiology and Infection School of Biosciences University of Birmingham Birmingham, United Kingdom Thomas E. Morrison, PhD Professor of Immunology and Microbiology University of Colorado School of Medicine Aurora, Colorado William J. Moss, MD, MPH Professor Department of Epidemiology Johns Hopkins Bloomberg School of Public Health Baltimore, Maryland Mark N. Namchuk, PhD Executive Director of Therapeutics Translation Professor of the Practice Biological Chemistry and Molecular Pharmacology Harvard Medical School Boston, Massachusetts Christopher J. Neufeldt, PhD Assistant Professor Department of Microbiology and Immunology Emory University Atlanta, Georgia Julie K. Pfeiffer, PhD Professor Department of Microbiology

University of Texas Southwestern Medical Center Dallas, Texas Shiv Pillai, MBBS, PhD Professor of Medicine Ragon Institute of MGH, MIT and Harvard Harvard Medical School Cambridge, Massachusetts James M. Pipas, PhD Herbert and Grace Boyer Chair of Molecular Biology Department of Biological Sciences University of Pittsburgh Pittsburgh, Pennsylvania John G. Purdy, PhD Associate Professor Immunobiology and Cancer Biology BIO5 Institute University of Arizona Tucson, Arizona Vincent R. Racaniello, PhD Higgins Professor Department of Microbiology & Immunology Vagelos College of Physicians and Surgeons Columbia University New York, New York John W. Schoggins, PhD Associate Professor Department of Microbiology UT Southwestern Medical Center Dallas, Texas Valerie L. Sim, MD, FRCPC Associate Professor Division of Neurology Department of Medicine Centre for Prions and Protein Folding Diseases University of Alberta Edmonton, Alberta, Canada Anne E. Simon, PhD Professor Department of Cell Biology and Molecular Genetics University of Maryland College Park, Maryland Gregory A. Storch, MD Ruth L. Siteman Professor of Pediatrics Washington University School of Medicine St. Louis, Missouri Jeanmarie Verchot, PhD Professor Plant Virology Plant Pathology & Microbiology Texas A&M University College Station, Texas David Wang, PhD Professor Molecular Microbiology and Pathology & Immunology Washington University in St. Louis St. Louis, Missouri

Sean P. J. Whelan, PhD Marvin A. Brennecke Distinguished Professor Chair, Molecular Microbiology School of Medicine Washington University in St. Louis St. Louis, Missouri Reed B. Wickner, MD NIH Distinguished Investigator Laboratory of Biochemistry & Genetics National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland Amy K. Winter, PhD Assistant Professor Department of Epidemiology and Biostatistics University of Georgia Athens, Georgia

Preface In the early 1980s, Bernie Fields originated the idea of a virology reference textbook that combined the molecular aspects of viral replication with the medical features of viral infections. This broad view of virology reflected Bernie’s own research, which applied molecular and genetic analyses to the study of viral pathogenesis, providing an important part of the foundation for the field of molecular pathogenesis. Bernie led the publication of the first three editions of Virology, but he unfortunately died soon after the third edition went into production. The third edition became Fields Virology in his memory, and it is fitting that the book continues to carry his name. A number of changes and enhancements have now been introduced with the seventh edition of Fields Virology. The publication format of Fields Virology has been changed from a once every 5-6 years, two volume book to an annual publication that comprises approximately one-fourth of the chapters organized by category. The annual publication provides both a physical book volume and importantly an eBook with an improved platform. Using an eBook format, our expectation is that individual chapters can be easily updated when major advances, outbreaks, etc., occur. The editorial board organized the four-volume series for the seventh edition to consist of volumes on Emerging Viruses, DNA Viruses, RNA Viruses, and Fundamental Virology, to be published on an annual basis, with the expectation that the topics will cycle approximately every 4 years creating an annualized, up-to-date publication. Each volume contains approximately 20 chapters. The first three volumes of this seventh edition of Fields Virology, entitled Emerging Viruses, DNA Viruses, and RNA Viruses were published in 2020, 2021, and 2022, respectively. This fourth volume, Fundamental Virology, has been principally edited by L. W. Enquist, Peter M. Howley, and David M. Knipe with valuable input from all of the Associate Editors. We wish to thank Patrick Waters of Harvard Medical School and all of the editorial staff members of Wolters Kluwer for all their important contributions to the preparation of this book. Peter M. Howley, MD David M. Knipe, PhD L. W. Enquist, PhD Jeffrey I. Cohen, MD Eric O. Freed, PhD Blossom Damania, PhD Sean P. J. Whelan, PhD

Introduction Fundamental Virology is the fourth volume of the seventh edition of Fields Virology. The first three volumes, Emerging Viruses, DNA Viruses, and RNA Viruses were published in 2020, 2021, and 2022, respectively. There have been continued rapid advances in virology since the sixth edition that was published in 2013, and all the chapters in this Fundamental Virology volume are either completely new or have been significantly updated to reflect these advances. Major advances in basic virology since the last edition have been included in this volume. This volume covers basic aspects of virology providing a historical context as well as fundamental principles, virus entry and uncoating, viral replication and assembly, and host immune responses to virology. There are chapters on viruses of insects, plants, bacteria, as well as chapters on prions as infectious agents. New chapters included in this seventh edition of Fields Virology are Metabolism and Viral Infection and Evolution of Viral Proteins. This Fundamental Virology volume is appropriate for use as a textbook for graduate students or advanced undergraduate students in virology courses, often when supplemented with individual virus chapters from Volumes 1-3 of this seventh edition. We are grateful to L. W. Enquist, Jeff I. Cohen, Blossom Damania, Eric O. Freed, and Sean P. J. Whelan who joined us as the senior editors and participated in putting this volume together. We also are thankful to the chapter authors who have updated their chapters for this volume and to the new authors who have joined us in this continued endeavor to provide a comprehensive resource in basic virology. Peter M. Howley David M. Knipe

Contents Contributors Preface Introduction

1 Virology: From Contagium Fluidum to Virome L. W. Enquist • Vincent R. Racaniello 2 Principles of Virology Julie K. Pfeiffer • Richard C. Condit • John W. Schoggins 3 Principles of Virus Structure Stephen C. Harrison 4 Virus Entry and Uncoating Jason Mercer 5 Viral Replication Strategies Sean P. J. Whelan 6 Virus Assembly and Maturation Christopher J. Neufeldt • Eric Hunter 7 Metabolism and Viral Infection James C. Alwine • John G. Purdy 8 Pathogenesis of Viral Infection Thomas E. Morrison • Mark T. Heise 9 Innate Immunity to Viruses Akiko Iwasaki • John W. Schoggins • Sun Hur 10 The Adaptive Immune Response to Viruses Dennis R. Burton • Shiv Pillai • Young S. Hahn • Thomas J. Braciale 11 Tumor Virology Daniel DiMaio • James M. Pipas 12 Evolution of Viral Proteins Eugene V. Koonin • Mart Krupovic 13 Epidemiology Amy K. Winter • William J. Moss 14 Antiviral Agents Donald M. Coen • Mark N. Namchuk • Daniel R. Kuritzkes 15 Immunization Against Viral Diseases James E. Crowe Jr 16 Diagnostic Virology Alexander L. Greninger • David Wang • Gregory A. Storch • Keith R. Jerome 17 Giant Viruses

Jônatas Santos Abrahão 18 Plant Viruses Anne E. Simon • Kristiina Mäkinen • Yi Li • Jeanmarie Verchot 19 Insect Viruses Paul D. Friesen 20 Viruses and Prions of Yeasts, Fungi, and Protists Reed B. Wickner 21 Bacteriophages Graham F. Hatfull 22 Prions Byron Caughey • Valerie L. Sim Index

CHAPTER 1 Virology: From Contagium Fluidum to Virome L. W. Enquist • Vincent R. Racaniello The concept of viruses as particulate infectious agents The birth of virology Pathogen discovery, 1886–1903 Plant viruses and the chemical period: 1929–1956 Bacteriophages Early years: 1915–1940 Phages and the birth of molecular biology: 1938–1970 Developing the modern concept of virology Animal viruses Cell culture technology and discovery: 1898–1965 The molecular and cell biology Era of virology The role of animal viruses in understanding eukaryotic gene regulation Animal viruses and the recombinant DNA revolution Animal viruses and oncology Vaccines and antivirals Virology and the birth of immunology Emerging viruses Epidemiology of viral infections Host-virus interactions and viral pathogenesis The COVID-19 pandemic Lessons learned Past is prolog The future of virology A role for systems biology in virology Genomics and the predictive power of sequence analysis The virome: how many viruses are there and where are they? Pathogen discovery Virology has had a remarkable history. Even though humans did not realize viruses existed until the late 1880s, viral infections have shaped the history and evolution of life on the planet. Viruses are likely to infect all living organisms, even though some have not yet been discovered. Not only do viral infections shape the evolution of their hosts but their hosts also influence the evolution of viruses. The consequences of viral infections not only altered human history but also have powerful effects on the entire ecosystem. As a result, virologists have gone to extraordinary lengths to study, understand, and in some cases attempt to eradicate these agents. It is noteworthy that just as the initial discovery of viruses required new technology (porcelain filters), uncovering the amazing biology underlying viral infections has gone hand in hand with new technology developments. Indeed, virologists have elucidated new principles of life processes and have been leaders in promoting new directions in science. For example, many of the concepts and tools of molecular and cell biology have been derived from the study of viruses and their host cells. This chapter is an attempt to review selected portions of this history as it relates to the development of new concepts in virology. A compendium of milestones in virology history is found in Table 1.1. TABLE 1.1

Milestones in virology

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See also54 and A. Murphy’s, online review: The Foundations of Medical and Veterinary Virology: Discoverers and Discoveries, Inventors and inventions, developers and technologies. http://talk.ictvonline.org/files/ictv_documents//documents/633.aspx

Abbreviations: ASV, avocado sunblotch viroid; BMV, brome mosaic virus; HAART, highly active antiretroviral therapy; HIV, human immunodeficiency virus; HPV, human papillomavirus; TBSV, tomato bushy stunt virus; TMV, tobacco mosaic virus; TRSV tobacco ringspot virus; SV40, simian virus; FMDV, foot and mouth disease virus; WHO, World Health Organization; MHC, major histocompatibility complex; RSV, Rous sarcoma virus.

The Concept of Viruses as Particulate Infectious Agents A diverse microbial world of bacteria, fungi, and protozoa had been widely accepted by the last half of the 19th century. An early proponent of the germ theory of disease was the noted German anatomist Jacob Henle of Gottingen (the discoverer of Henle loop in kidneys and the grandfather of 20th-century virologist Werner Henle). He hypothesized in 1840 that specific diseases were caused by infectious agents that were 3

too small to be observed with the light microscope. But he had no evidence for such entities, and consequently his ideas were not generally accepted. It would take the work of Louis Pasteur and Henle’s student, Robert Koch, before it became evident that microbes could cause diseases. As early as 1728, the term virus was used to describe an agent that causes infectious disease. Long before bacteria, fungi, and viruses were identified, it was known that some diseases could be transmitted from person to person. The nature of such agents was unknown, but because the word virus comes from the Latin meaning a poison, we can surmise that the agents were thought to be liquids. In the 1800s, the work of Pasteur, Lister, and Koch founded the field of microbiology, leading to the germ theory of disease: microbes could cause infectious diseases. Louis Pasteur (1822–1895) studied fermentation by different microbial agents. From his work, he concluded that “different kinds of microbes are associated with different kinds of fermentations” and he soon extended this concept to diseases. In fact, Pasteur thought that all infectious diseases were caused by microbes, as suggested by his statement that “every virus is a microbe.” Pasteur’s reasoning strongly influenced Robert Koch (1843–1910), a student of Jacob Henle and a country doctor in a small German village. He developed solid media to isolate colonies of bacteria to produce pure cultures and stains to visualize the microorganisms. With these tools in hand, he identified the bacterium that causes anthrax (Bacillus anthracis, 1876) and tuberculosis (Mycobacterium tuberculosis, 1882). Joseph Lister (1827–1912), a professor of surgery in Glasgow, had heard about Pasteur’s work and surmised that a sterile field should be maintained during surgery. Although many other scientists of that day contributed tools and concepts, it was principally Pasteur, Lister, and Koch who put together a new experimental approach for medical science. These observations led Robert Koch to formalize some of Jacob Henle’s original ideas for defining whether a microorganism is the causative agent of a disease. Koch’s postulates state that (1) the organism must be regularly found in the lesions of the disease, (2) the organism must be isolated in pure culture, (3) inoculation of such a pure culture of organisms into a host should initiate the disease, and (4) the organism must be recovered once again from the lesions of the host. By the end of the 19th century, these concepts outlined an experimental method that became the dominant paradigm of medical microbiology. It was only when these rules broke down and failed to yield a causative agent that the concept of a virus as an infectious particle was born. A key point in the history of virology was the development in 1884 by Charles Chamberland, an associate of Pasteur, of a porcelain filter that could retain bacteria. The filters were initially used to provide bacteria-free water for the laboratory. Pasteur, working on the agent of rabies, found that it could pass through such filters and thought it to be unusually small. Later Ivanovsky and Beijerinck showed that the agent of tobacco mosaic disease to be small enough to pass through filters. Beijerinck had the important insight that the pathogen could only be propagated in plants not in broth as was known for bacteria. He called it a “contagious living fluid.” After this work, many similar agents were found and the name “filterable virus” was applied to distinguish them from infectious agents that were retained on the filter. At this point, the distinctions between bacteria and viruses were on the basis of filterability and reproduction in broth. The exact nature of “filterable viruses” was obscure. Four subsequent observations were essential in leading to our current concept of viruses. Bacteriophages were discovered in 1917 and found to cause clear spots, or plaques, on lawns of bacteria growing on agar plates. Such behavior did not fit in with the previously “fluid” nature of viruses. Bacteriophages were visualized by the electron microscope for the first time in 1939, proving beyond all doubt that they were particulate. Crystals of the tobacco mosaic disease agent were produced by Stanley in 1935, which to chemists proved that filterable viruses were not only chemicals but could be made pure and homogeneous. To Stanley, this observation meant that filterable viruses were “infectious proteins,” a conclusion he reached by ignoring the small amounts of phosphate present in his crystals. He was wrong about tobacco mosaic virus (TMV), whose RNA was later shown to be infectious, but many years later infectious proteins (prions) were discovered. The last important observation on the road to virus was the development of the one-step growth curve of bacteriophages by Ellis and Delbruck in 1939. They noted that when viruses were added to bacteria, there was initially a lag phase during which no new infectious viruses were produced. This lag phase contrasted with observations made with bacteria: when added to broth, they immediately began to divide. This result showed that viruses were not simply miniature bacteria. We now know that the lag phase is a period during which new viral components are being synthesized, which are later assembled to form infectious particles. Bacteria reproduce by binary fission, and viruses reproduce by making new parts and assembling them. As a consequence of these observations, the word “filterable” was dropped and these agents became simply “viruses” as their distinction from bacteria and other infectious agents became clear. And that is how we came to today’s definition of virus, which began as a catchall for all agents of infectious diseases.

The Birth of Virology

Pathogen Discovery, 1886–1903 Adolf Mayer (1843–1942) was a German agricultural chemist and director of the Agricultural Experiment Station at Wageningen in The Netherlands when he was asked to investigate a disease of tobacco. He named the affliction tobacco mosaic disease after the dark and light spots that appeared on infected leaves (Fig. 1.1). To investigate the nature of the disease, Mayer inoculated healthy plants with the juice extracted from diseased plants by grinding up the infected leaves in water. Mayer reported that, “in nine cases out of ten (of inoculated plants), one will be successful in making the healthy plant . . . heavily diseased”.122 Although these studies established the infectious nature of the tobacco mosaic disease, neither a bacterial nor a fungal agent could consistently be cultured or detected in these extracts, so Koch’s postulates could not be satisfied. In a preliminary communication in 1882,121 Mayer speculated that the cause could be a “soluble, possibly enzymelike contagium, although almost any analogy for such a supposition is failing in science.” Later Mayer concluded that the mosaic disease “is bacterial, but that the infectious forms have not yet been isolated, nor are their forms and mode of life known”.122

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FIGURE 1.1 Lesions on a tobacco leaf infected with tobacco mosaic virus. Adolph Mayer was the first to describe the pattern of light and dark green areas on infected leaves in 1886. Mayer showed that lesions could be transmitted from one plant to another using extracts produced from an infected leaf. A few years later, Dimitri Ivanofsky (1864–1920), a Russian scientist working in St. Petersburg, was commissioned by the Russian Department of Agriculture to investigate the cause of a tobacco disease on plantations in Bassarabia, Ukraine, and the Crimea. Ivanofsky repeated Mayer’s observations by showing that the sap of infected plants contained an agent that could transmit the disease to healthy plants. But he added an important step—before the inoculation step, he passed the infected sap through a Chamberland filter (Fig. 1.2). This device, made of unglazed porcelain and perfected by Charles Chamberland, one of Pasteur’s collaborators, contained pores small enough to retard most bacteria. Ivanofsky reported to the Academy of Sciences of St. Petersburg on February 12, 1892 that “the sap of leaves infected with tobacco mosaic disease retains its infectious properties even after filtration through Chamberland filter candles”.90

FIGURE 1.2 Chamberland filter. These filters, first used to demonstrate the filterable nature of viruses, are made of diatomaceous earth pressed into the form of a hollow candle. The fluid to be filtered is placed into the opening at the top using a funnel, and the filtrate, containing viruses, is collected in the flask. Ivanofsky, like Mayer before him, failed to culture an organism from the filtered sap and could not satisfy Koch’s postulates. Consequently, he suggested that a toxin (not a living, reproducing substance) might pass through the filter and cause the disease. As late as 1903, when Ivanofsky published his thesis,91 he still believed that he had been unable to culture the bacteria that caused this disease. Bound by the dogma of Koch’s postulates, Ivanofsky could not make a conceptual leap. It is, therefore, not surprising that Pasteur, who worked on the rabies vaccine135 at the same time (1885), never investigated the unique nature of the infectious agent.

The conceptual leap was provided by Martinus Beijerinck (1851–1931), a Dutch soil microbiologist who collaborated with Adolf Mayer at Wageningen. Unaware of Ivanofsky’s work, in 1898, he independently found that the sap of infected tobacco plants could retain its infectivity after passage through a Chamberland filter. But he also showed that the filtered sap could be diluted and regain its “strength” after replication in living, growing tissue of the plant. This observation showed that the agent could reproduce (therefore, it was not a toxin) but only in living tissue, not in the cell-free sap of the plant. Suddenly it became clear why others could not culture the pathogen outside its host. Beijerinck called this agent a contagium vivum fluidum,10 or a contagious living liquid. He sparked a 25-year debate, summarized above, about whether these novel agents were liquids or particles. This conflict was resolved when d’Herelle developed the plaque assay in 191733 and when the first electron micrographs were taken of TMV in 1939.99

Mayer, Ivanofsky, and Beijerinck each contributed to the development of a new concept: a novel organism smaller than bacteria—an agent defined by the pore size of the Chamberland filter—which could not be seen in the light microscope and could multiply only in living cells or tissue. The term virus (from the Latin for slimy liquid or poison85) was at that time used interchangeably for any infectious agent, and so the agent of tobacco mosaic disease was called tobacco mosaic virus, TMV. The literature of the first decades of the 20th century often referred to these infectious entities as filterable agents, and this was indeed the operational definition of viruses. Sometime later, the term virus became restricted in use to those agents that fulfilled the criteria developed by Mayer, Ivanofsky, and Beijerinck. 5

Shortly after this pioneering work on TMV, the first filterable agent from animals was identified by Loeffler and Frosch—foot and mouth disease virus.113 The first human virus discovered was yellow fever virus (1901), by Walter Reed and his team in Cuba.144

Plant Viruses and the Chemical Period: 1929–1956 For the next 50 years, TMV played a central role in research that explored the nature and properties of viruses. With the development of techniques to purify proteins in the first decades of the 20th century came the appreciation that the infectious material that passed through filters was proteins and so could be purified in the same way. Working at the Boyce Thompson Institute in Philadelphia, Vinson and Petre (1927–1931) precipitated infectious TMV (using an infectivity assay developed by Holmes84) from the crude sap of infected plants using selected salts, acetone, or ethyl alcohol.178 They showed that the infectious material could move in an electric field, just as proteins did. At the same time, H. A. Purdy-Beale, also at the Boyce Thompson Institute, produced antibodies in rabbits that were directed against TMV and could neutralize the infectivity of this agent.140 This observation was taken as further proof of the protein nature of viruses, although it was later realized that antibodies recognize chemicals other than proteins. With the advent of purification procedures for viruses, both physical and chemical measurements became possible. The strong flow birefringence of purified preparations of TMV was interpreted (correctly) to show an asymmetric particle or rod-shaped particle.165 Max Schlesinger,152 working on purified preparations of bacteriophages in Frankfurt, Germany, showed that the infectious particles were composed of proteins and also contained phosphorus and ribonucleic acid. This observation led to the first suggestion that virus particles were composed of nucleoproteins. The crystallization of TMV in 1935 by Wendell Stanley,158 working at the Rockefeller Institute branch in Princeton, New Jersey, brought this infectious agent into the world of the chemists. Within a year, Bawden and Pirie8,9 had demonstrated that crystals of TMV contained 0.5% phosphorus and 5% RNA. The first “view” of a virus came from x-ray crystallography using these crystals to show rods of a constant diameter aligned in hexagonal arrays containing RNA and protein.16 The first electron micrographs of any virus were of TMV, and they confirmed that the virus particle is shaped like a rod99 (Fig. 1.3).

FIGURE 1.3 Electron micrograph of the tobacco mosaic virus particle. The virion is composed of a naked, helical nucleocapsid composed of multiple copies of a single viral protein complexed with the viral RNA genome. The x-ray diffraction patterns16 suggested that TMV was built from repeating subunits. These data and other considerations led Crick and Watson32 to realize that most simple virus particles had to consist of one or a few species of identical protein subunits. By 1954–1955, techniques had been developed to dissociate TMV protein subunits, allowing reconstitution of infectious TMV from its RNA and protein subunits63 and leading to an understanding of the principles of virus self-assembly.25

The concept that virus particles contained genetic information emerged as early as 1926, when H. H. McKinney reported the isolation of “variants” of TMV with a different plaque morphology that bred true and could be isolated from several geographic locations.123,124 Seven years later, Jensen confirmed McKinney’s observations96 and showed that the plaque morphology phenotype could revert. Avery’s DNA transformation experiments with pneumococcus96 and the Hershey-Chase experiment with bacteriophages80 both demonstrated that DNA was genetic material. TMV had been shown to contain RNA, not DNA, and in 1956, this nucleic acid was shown to be infectious. RNA, therefore, comprised the genetic material of the virus and was the first demonstration that RNA could be a genetic material.63,70 Studies on the nucleotide sequence of TMV RNA confirmed codon assignments for the genetic code, adding clear evidence for the universality of the genetic code. These studies helped to elucidate the mechanisms of mutation by diverse agents.62 Research on TMV and related plant viruses has contributed significantly to both the origins of virology and its development as a science.

Bacteriophages

Early Years: 1915–1940 Frederick W. Twort was superintendent of the Brown Institution in London when he discovered viruses of bacteria in 1915. In his research, Twort was searching for variants of vaccinia virus (the smallpox vaccine virus) that would replicate in simple defined media outside living cells. In one of his experiments, he inoculated nutrient agar with an aliquot of the smallpox vaccine (which essentially was lymph fluid from vaccinia pustules). The virus failed to replicate, but bacterial contaminants flourished on the agar medium. Twort noticed that some of these bacterial colonies changed visibly with time and became “watery looking” (ie, more transparent). The bacteria within these colonies were apparently dead, as they could no longer form new colonies on fresh agar plates. He called this phenomenon “glassy transformation.” Simply adding the glassy transforming principle could rapidly kill a colony of bacteria. It readily passed through a porcelain filter, could be diluted a million-fold, and when placed upon fresh bacteria would regain its strength, or titer.173–175

Twort published these observations in a short note173 in which he suggested that a virus of bacteria could explain glassy transformation. He then went off to serve in World War I, and when he returned to London, he did not continue this research.

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While Twort was puzzled by glassy transformation, Felix d’Herelle, a Canadian medical bacteriologist, was working at the Pasteur Institute in Paris. When a Shigella dysentery infection devastated a cavalry squadron of French soldiers just outside of Paris in August 1915, D’Herelle readily isolated and cultured the dysentery bacillus from filtered fecal emulsions. The bacteria multiplied and covered the surface of his agar plates, but occasionally d’Herelle observed clear circular spots devoid of growth. He called these areas taches vierges, or plaques. He followed the course of an infection in a single patient, noting when the bacteria were most plentiful and when the plaques appeared.33,34 Plaques appeared on the fourth day after infection and killed the bacteria in the culture dish, after which the patient’s condition began to improve.

D’Herelle found that a filterable agent, which he called a bacteriophage, was killing the Shigella bacillus. In the ensuing years, he developed fundamental techniques in virology that are utilized to this day, such as the use of limiting dilutions to determine the virus titer by plaque assay. He reasoned that the appearance of plaques showed that the virus was particulate, or “corpuscular,” and not a liquid as Beijerinck had insisted. D’Herelle also found that if infectious material was mixed with a host cell and then subjected to centrifugation, the infectious material was no longer present in the supernatant fluid. He interpreted this to mean that the first step of a virus infection is attachment, or adsorption, of virus particles to the host cell. Furthermore, particle attachment occurred only when bacteria sensitive to infection were used, demonstrating that host specificity can be conferred at a very early step in infection. Lysis of cells and the release of infectious particles were also described in startlingly modern terms. D’Herelle clearly established many of the principles of modern virology.34,35

Although D’Herelle’s bacteriophages lysed their host cells, by 1921 it had become apparent that under certain situations the bacteriophage and cell existed peacefully—a condition called lysogeny. In some experiments, it became impossible to separate the virus from its host. This condundrum led Jules Bordet of the Pasteur Institute in Brussels to suggest that the transmissible agent described by d’Herelle was nothing more than a bacterial enzyme that stimulates its own production.22 Although incorrect, the hypothesis has remarkable similarities to modern ideas about prion structure and replication (see Chapter 22).

During the 1920s and 1930s, d’Herelle sought ways to use bacteriophages for medical applications, but he never succeeded. Interestingly, phage therapy (the use of bacteriophages to treat patients with bacterial infections) has seen renewed interest. To complicate matters, the basic research of the era was dominated by the interpretations of scientists with the strongest personalities. Although it was clear that there were many diverse bacteriophages, and that some were lytic while some were lysogenic, their interrelationships remained ill defined and contentious. The highlight of this period was the demonstration by Max Schlesinger that purified phages had a maximum linear dimension of 0.1 micron and a mass of about 4 × 10−16 g, and that they were composed of protein and DNA in roughly equal proportions.151,152 In 1936, no one quite knew what to make of that observation, but over the next 20 years, it would begin to make a great deal of sense.

Phages and the Birth of Molecular Biology: 1938–1970 Max Delbrück was trained as a physicist at the University of Göttingen, and his first position was at the Kaiser Wilhelm Institute for Chemistry in Berlin. There he joined a diverse group of individuals actively discussing how quantum physics related to an understanding of heredity. Delbrück’s interest in this area led him to develop a quantum mechanical model of the gene, and in 1937, he moved to the Biology Division at the California Institute of Technology to study genetics of Drosophila. Once there, he became interested in bacteria and their viruses and teamed up with another research fellow, Emory Ellis,50 who was working with the T-even group of bacteriophages, T2, T4, and T6. Delbrück soon appreciated that these viruses were ideal for the study of how genes replicated and how genetic information could determine the structure and function of an organism. They replicated rapidly and offered simple quantitative assays. Delbruck brought the rigor of quantitative, well-defined experiments to virology. Bacteriophages were also viewed as model systems for understanding cancer viruses or even for understanding how a sperm fertilizes an egg and a new organism develops. Together with Ellis, Delbrück showed that when infections were synchronized, new virus particles were produced in one step (a burst),51 in contrast to the multiplication of other organisms by binary fission. The elegant one-step growth curve experiment showed that a single infected bacterium liberates hundreds of phages synchronously after a half hour period during which viral infectivity was lost (Fig. 1.4). The one-step growth curve became the experimental paradigm of the phage group.

FIGURE 1.4 One-step growth curve of bacteriophage. The conclusion of Ellis and Delbrück that viruses reproduced in one step was drawn from the one-step growth curve experiment, in which an infected bacterium liberates phages synchronously after a half hour period (eclipse) during which viral infectivity is lost. When World War II erupted, Delbrück remained in the United States (at Vanderbilt University) and met an Italian refugee, Salvador E. Luria, who had fled to America and was working at Columbia University in New York (on bacteriophages T1 and T2). After their encounter at a meeting in Philadelphia on December 28, 1940, they went to Luria’s laboratory at Columbia where they spent 48 hours doing experiments with bacteriophages. These two scientists eventually established the “phage group,” a community of researchers focused on using bacterial viruses as a model for understanding life processes. Luria and Delbruck were invited to spend the summer of 1941 at Cold Spring Harbor Laboratory, where they pursued research on phages. It is remarkable that a German physicist and an Italian geneticist joined forces during the war years to travel throughout the United States and recruit a new generation of biologists (Fig. 1.5).

7

FIGURE 1.5 Max Delbrück and Salvador Luria at Cold Spring Harbor Laboratory. Photograph taken during the 1953 Cold Spring Harbor Symposium on Quantitative Biology organized by Milislav Demerec. (From http://profiles.nlm.nih.gov/ps/retrieve /ResourceMetadata/QLBBJF. Courtesy of the Cold Spring Harbor Laboratory Archives, NY.) When Tom Anderson, an electron microscopist at the RCA Laboratories in Princeton, New Jersey, met Delbrück, the result was the first clear pictures of bacteriophages.117 At the same time, the first phage mutants were isolated and characterized.115 By 1946, the first phage course was being taught at Cold Spring Harbor, and in March 1947, the first phage meeting attracted eight people. From these humble beginnings, the field of molecular biology emerged, which focused on the bacterial host and its viruses.

Developing the Modern Concept of Virology The next 25 years (1950–1975) was an intensely productive period of bacteriophage research. Hundreds of virologists produced thousands of publications that covered three major areas: (1) lytic infection of Escherichia coli with the T-even phages; (2) the nature of lysogeny, using lambda phage; and (3) the replication and properties of several unique phages such as ϕX174 (single-stranded circular DNA), the RNA phages, and T7. This work set the foundations for modern molecular virology and biology. The idea of examining, at the biochemical level, the events occurring in phage-infected cells during the latent period had come into its own by 1947–1948. Impetus for this work came from Seymour Cohen, who had trained first with Erwin Chargaff at Columbia University, studying lipids and nucleic acids, and then with Wendell Stanley working on TMV RNA. His research direction was established after taking Delbrück’s 1946 phage course at Cold Spring Harbor, Cohen examined the effects of phage infection on DNA and RNA levels in infected cells using a colorimetric analysis.30 The results showed a dramatic alteration of macromolecular synthesis in infected cells. This included cessation of RNA accumulation, which later formed the basis for detecting a rapidly turning over species of RNA and the first demonstration of messenger RNA (mRNA).4 DNA synthesis also halted, but only for 7 minutes, followed by resumption at a 5- to 10-fold increased rate. At the same time, Monod and Wollman showed that the synthesis of a cellular enzyme, the inducible β-galactosidase, was inhibited after phage infection.125 Based on these observations, the viral eclipse period (when infectivity was lost immediately after infection) was divided into an early phase, prior to DNA synthesis, and a late phase. More importantly, these results demonstrated that virus infection could redirect cellular macromolecular synthetic processes in infected cells.31

By the end of 1952, two experiments had a critical effect on virology. First, Hershey and Chase asked whether bacteriophage genetic information is DNA or protein. They differentially labeled viral proteins (with 35SO4) and nucleic acids (with 32PO4) and allowed the radioactive “tagged” particles to attach to bacteria. When they sheared the viral protein coats from the bacteria using a Waring blender, only DNA was associated with the infected cells.80 This result proved that DNA had all the information needed to reproduce new virus particles. A year later, the structure of DNA was elucidated by Watson and Crick, a discovery that permitted full appreciation of the Hershey-Chase experiment.180 The results of these two experiments formed a cornerstone of the molecular biology revolution.

While these blockbuster experiments were being carried out, G. R. Wyatt and S. S. Cohen were quietly making another seminal finding.191 They identified a new base, hydroxymethylcytosine, in the DNA of T-even phages, which replaced cytosine. This began a 10-year study of how deoxyribonucleotides were synthesized in bacteria and phage-infected cells and led to the critical observation that bacteriophage infection introduces genetic information for a new enzyme.59 By 1964, Mathews and colleagues had proved that hydroxymethylase does not exist in uninfected cells and must be encoded by the virus genetic material.31 These experiments introduced the concept of early enzymes, utilized in deoxypyrimidine biosynthesis and DNA replication,102 and provided biochemical proof that viral genomes encoded new information expressed as proteins in an infected cell. At the same time, phage genetics became extremely sophisticated, allowing mapping of the genes encoding these viral proteins. Perhaps the best example of genetic fine structure was done by Seymour Benzer, who carried out a genetic analysis of the rII A and B cistrons of T-even phages with a resolution of a single nucleotide (without doing any DNA sequencing!).13 Studies on viral DNA synthesis, using phage mutants and cell extracts to complement and purify enzyme activities in vitro, contributed a great deal to our understanding of DNA replication.1 A detailed genetic analysis of phage assembly, utilizing the complementation of phage assembly mutants in vitro, revealed 8

how complex structures are built using the principles of self-assembly.45 The genetic and biochemical analysis of phage lysozyme helped to elucidate the molecular nature of mutations,161 and the isolation of phage nonsense mutations (first called “amber” mutations) provided a clear way to study second-site suppressor mutations at the molecular level.14 The circular genetic map of the T-even phages161 was explained by the circularly permuted, terminally redundant conformation of these DNAs (giving rise to phage heterozygotes).171

The remarkable reprogramming of viral and cellular protein synthesis in phage-infected cells was dramatically visualized by an early use of electrophoresis and sodium dodecyl sulfate (SDS)-polyacrylamide gels.104 These studies showed that viral proteins are made in a specific sequence of events. The underlying mechanism of this temporal regulation led to the discovery of proteins (sigma factors) that modified RNA polymerase and conferred specificity to gene expression.73 The study of gene regulation at almost every level (transcription, RNA stability, protein synthesis, protein processing) was revealed from a set of original contributions derived from an analysis of phage infections.

Although this remarkable progress had begun with the lytic phages, no one knew quite what to make of the lysogenic phages. This situation changed in 1949 when André Lwoff began his studies at the Pasteur Institute with Bacillus megaterium and its lysogenic phages. By using a micromanipulator, Lwoff could show that single lysogenic bacteria divided up to 19 times before liberating infectious particles. No virions were detected when lysogenic bacteria were broken open by the investigator. But from time to time, a lysogenic bacterium spontaneously lysed and produced many infectious particles.119 Many potential inducing agents were investigated, but ultraviolet light was very efficient. Indeed, exposing lysogenic bacteria to ultraviolet light efficiently induced production of infectious particles. This experiment began120 to outline this curious relationship between a virus and its host. By 1954, Jacob and Wollman93,94 at the Pasteur Institute had made the important observation that a genetic cross between a male, lysogenic bacterial strain (Hfr, lambda) and a nonlysogenic female recipient resulted in the induction of infectious particles after conjugation, a process they called zygotic induction. In fact, the position of the lysogenic phage (also called prophage) in the chromosome of its host E coli could be mapped by interrupting mating between two strains.94 This experiment was crucial for our understanding of lysogenic viruses because it showed that the provirus behaved like a bacterial gene on a chromosome in a bacterium. It also was one of the first experimental results suggesting that the prophage genetic material was kept quiescent by negative regulation, which was lost as the chromosome passed from the lysogenic donor bacteria to the nonlysogenic recipient host. This conclusion helped Jacob and Monod to realize as early as 1954 that the “induction of enzyme synthesis and of phage development are the expression of one and the same phenomenon”.119 These experiments laid the foundation for the operon model and180 the nature of coordinate gene regulation.

Although the structure of DNA was elucidated in 1953 and zygotic induction was described in 1954, the physical interaction between the bacterial chromosome and the viral genetic material in lysogeny was not clear. It often was referred to as the “attachment site” and literally thought of in those terms. The close relationship between a virus and its host was appreciated only when Campbell proposed the model for physical integration of lambda DNA into the bacterial chromosome,26 based on the fact that the sequence of phage markers was different in the integrated state than in the replicative or vegetative state. The idea was that a circular lambda DNA molecule integrated by recombination between a specific site on the phage DNA with a specific site on the bacterial DNA. This integrated DNA was the prophage, which was transcriptionally silent. This model led to the isolation of the negative regulator or repressor of lambda, the understanding of why lysogens could not be infected by related bacteriophages (so-called immunity), and one of the early examples coordinated gene regulation.139 The genetic analysis of the lambda bacteriophage life cycle is one of the great intellectual adventures in microbial genetics.79 It deserves to be reviewed in detail by all students of molecular virology and biology. Other lysogenic E coli phages offered different paradigms that did not involve integration of viral DNA into host DNA. Bacteriophage P1 has a circular genome that is maintained in the cytoplasm as an episome and maintains stable inheritance by repressors and cytoplasmic replication.

The lysogenic phages193 of Salmonella typhimurium such as P22 provided the first example of generalized transduction, whereas lambda provided the first example of specialized transduction.128 The finding that virus particles could not only carry cellular genes but transfer those genes from one cell to another provided not only a method for fine genetic mapping but also a new concept in virology. As the genetic elements of bacteria were studied in more detail, it became clear that there was a remarkable continuum from lysogenic phages to episomes, transposons and retrotransposons, insertion elements, retroviruses, hepadnaviruses, viroids, and prions. Genetic information moves between viruses and their hosts to the point where definitions and classifications begin to blur.

The genetic and biochemical concepts that emerged from the study of bacteriophages stimulated the next phase of virology. The lessons of the lytic and lysogenic phages were often relearned and modified as the animal viruses were studied.

Animal Viruses

Cell Culture Technology and Discovery: 1898–1965 Once the concept took hold that viruses were particulate filterable agents, many diseased animal tissues were subjected to filtration to determine if a virus were involved. Filterable agents were found that were invisible in a light microscope and replicated only in living animal tissue. There were some surprises, such as the transmission of yellow fever virus by a mosquito vector,144 specific visible pathologic inclusion bodies (virions and subviral particles) in infected tissue,91,133 and even viral agents that can “cause cancer.”49,146 Throughout this early period (1900–1930), a wide variety of particulate animal infectious agents were found (see Table 1.1) and characterized with regard to their size (using the different pore sizes of filters), resistance to chemical or physical agents (eg, alcohol, ether), and pathogenic effects. Just based on these properties, it became clear that viruses were a very diverse group. Some were even observable in the light microscope (vaccinia in dark-field optics). Some were inactivated by ether, whereas others were not. Infectious agents that replicated in every tissue type were identified. Some were specific for a particular tissue. They could cause chronic or acute disease; they were persistent agents or recurred in a periodic fashion. Some caused cellular destruction or induced cellular proliferation. Some changed the morphology of the cells they infected. For the early virologists, unable to see their agents in a light microscope and often confused by this great diversity, many studies certainly required an element of faith. In 1912, S. B. Wolbach, an American pathologist, remarked, “It is quite possible that when our knowledge of filterable viruses is more complete, our conception of living matter will change considerably, and that we shall cease to attempt to classify the filterable viruses as animal or plant”.188

The first way out of this early confusion was led by the plant virologists and the development of techniques to purify viruses and characterize both the chemical and physical properties of these agents (see previous section, The Plant Viruses and the Chemical Period: 1929–1956). The second path came from the studies with bacteriophages, where single cells infected with viruses in culture were much more amenable to experimental manipulation than were virus infections of whole animals. Whereas the plant virologists of that day were tethered to their 9

greenhouses, and the animal virologists were bound to their animal facilities, the viruses of bacteria were studied in Petri dishes and test tubes. Nevertheless, progress was made in the study of animal viruses one step at a time; from studying animals in the wild, to laboratory animals, such as the mouse64 or embryonated chicken eggs,189 to the culture of tissue, and then to single cells. Between 1948 and 1955, a critical transition converting animal virology into a laboratory science came in four important steps: Sanford and colleagues at the National Institutes of Health (NIH) overcame the difficulty of culturing single cells149; George Gey at Johns Hopkins Medical School cultured and passaged human cells for the first time and developed a line of immortal cells (HeLa) from a cervical carcinoma69; and Harry Eagle at the NIH developed an optimal medium for the culture of single cells.44 In a demonstration of the utility of all these methods, Enders and his colleagues showed that poliovirus could replicate in a nonneuronal human explant of embryonic tissues.53

These ideas, technical achievements, and experimental advances had immediate effects on the field of virology. One of these was the development of the polio vaccine, the first ever produced in cell culture. From 1798 to 1949, all the vaccines in use (smallpox, rabies, yellow fever, influenza) had been grown in animals or embryonated chicken eggs. Poliovirus was grown in monkey kidney cells propagated in flasks.81,110 The modern era of molecular virology began with the exploitation of cell culture. The first plaque assay for an animal virus in culture was done with poliovirus,41 and it led to an analysis of poliovirus every bit as detailed and important as the contemporary work with bacteriophages. The simplest way to document this statement is for the reader to compare the first edition of General Virology by S. E. Luria in 1953116 to the second edition by Luria and J. E. Darnell in 1967118 and to examine the experimental descriptions of poliovirus infection of cells. The modern era of virology had arrived, and it would continue to be full of surprises.

The Molecular and Cell Biology Era of Virology The history of virology has so far been presented chronologically or according to separate virus groups (plant viruses, bacteriophages, and animal viruses), which reflects the historical separation of these fields. In this section, the format changes as the motivation for studying viruses began to change. Virologists began to use viruses to probe questions central to understanding all life processes. Because viruses replicate in and are dependent on their host cells, they must use the rules, signals, and regulatory pathways of the host. By using viruses to probe these processes, virologists began to make contributions to all facets of biology. This approach began with the phage group and was continued by the animal virologists. The recombinant DNA revolution also took place during this period (1970 to the present), and both bacteriophages and animal viruses played a critical and central role in this revolution. For these reasons, the organization of this section focuses on the advances in cellular and molecular biology made possible by experiments with viruses. Some of the landmarks in virology since 1970 are listed in Table 1.1.

The Role of Animal Viruses in Understanding Eukaryotic Gene Regulation Viral genetic material can be DNA or RNA, single stranded or double stranded.156 How these forms of nucleic acid are replicated, how gene products are produced, and how they are packaged into infectious particles were and still are fundamental questions. The closed circular and superhelical nature of polyoma virus DNA was elucidated by Dulbecco and Vogt42 and Weil and Vinograd.181 The reason why the viral genome adopted this structure was first shown to be related to the packaging of simian vacuolating virus 40 (SV40) DNA into virus particles. The viral DNA is wound around nucleosomes during replication and is packaged into particles.68 When the histones are removed, a DNA superhelix results. The structure of polyoma viral DNA served as an excellent model for the E coli genome190 and mammalian chromosomes.105,106

Many elements of the eukaryotic transcription machinery have been elucidated with viruses. The first transcriptional enhancer element (acts in an orientation- and distance-independent fashion) was described in the SV40 genome,74 as was a distance- and orientation-dependent promoter element.108 The transcription factors that bind to the promoter, SP-1,43 or to the enhancer element, such as AP-1 and AP-2, and which are essential to promote transcription along with the basal factors, were first described with SV40. AP-1 is composed of fos and jun family member proteins, demonstrating the role of transcription factors as oncogenes.21 Indeed, the great majority of experimental data obtained for basal and accessory transcription factors come from in vitro transcription systems using the adenovirus major late promoter or the SV40 early enhancer-promoter.182 Our present-day understanding of RNA polymerase III promoter recognition comes, in part, from an analysis of the adenovirus VA gene transcribed by this polymerase.61

Almost everything we know about the steps of mRNA processing began with observations made with viruses. The list is long but here are a few highlights: RNA splicing of new transcripts was first described in adenovirus-infected cells15,28; polyadenylation of mRNA was first observed with poxviruses,97 the first viruses shown to have a DNA-dependent RNA polymerase in the virion98; the signal for polyadenylation of mRNA was identified using SV4058; the methylated cap structure found at the 5’-end of most mRNAs was first discovered on reovirus mRNAs65; and a remarkable discrimination for transport of viral and cellular mRNAs out of the nucleus was shown by the adenovirus E1B-55Kd protein.137

Our early understanding of translational regulation came from studies of virus infected cells. Recruitment of ribosomes to mRNAs was shown to be directed by the 5’-cap structure first discovered on reovirus mRNAs. The nature of the protein complex that allows ribosomes to bind the 5’cap was elucidated in poliovirus-infected cells, because viral infection leads to cleavage of one of the components, eIF4G. Internal initiation of translation was discovered in cells infected with picornaviruses (poliovirus and encephalomyocarditis virus).95,136 Interferon, discovered as a set of proteins that inhibited influenza virus replication, was subsequently found to induce the synthesis of many antiviral gene products that act on translational regulatory processes.88 Similarly, the viral defenses against interferon by the adenovirus VA RNA provided unique insight to the role of eIF-2 phosphorylation events.101 Mechanisms for producing more than one protein from a eukaryotic mRNA (there is no “one mRNA one protein” rule as described for bacteria) were discovered in virus-infected cells, including polyprotein synthesis, ribosomal frameshifting, and leaky scanning. Posttranslational processing of proteins by proteases, carbohydrate addition to proteins in the Golgi apparatus, phosphorylation by a wide variety of important cellular protein kinases, or the addition of fatty acids to membrane-associated proteins have all been profitably studied using viruses. Indeed, a good deal of our present-day knowledge of how protein trafficking occurs and is regulated in cells comes from the use of virus-infected cell systems. The field of gene regulation has derived many of its central tenets from the study of viruses.

Animal Viruses and the Recombinant DNA Revolution The discovery of the enzyme reverse transcriptase5,170 not only elucidated the replication cycle of retroviruses but also provided an essential 10

tool to convert RNA molecules to DNA, which could then be cloned and manipulated. The first restriction enzyme map of a chromosome was done with SV40 DNA, using the restriction enzymes HindII plus HindIII DNA.36,37 The first demonstration of restriction enzyme specificity was carried out with the same viral DNA cleaved with EcoRI.127,129 Some of the earliest DNA cloning experiments involved insertion of SV40 DNA into bacteriophage lambda DNA, or human β-hemoglobin genes into SV40 DNA, yielding the first mammalian expression vectors.92 A debate about whether these very experiments were potentially dangerous led to a temporary moratorium on all such recombinant experiments following the scientist-organized Asilomar Conference. From the earliest experiments in the field of recombinant DNA, several animal viruses had been developed into expression vectors to carry foreign genes, including SV40,72 retroviruses,183 adenoviruses,67 and adeno-associated virus.148 Modern day strategies of gene therapy rely on some of these recombinant viruses. The gene for beta hemoglobin was first cloned using lambda vectors. The elusive hepatitis virus C (previously known as non-A, non-B) viral genome was cloned from serum using recombinant DNA techniques, reverse transcriptase, and lambda phage vectors.27

The study of RNA viruses was accelerated by recombinant DNA technology, particularly in the construction of “infectious clones.” The RNA virus genome was converted into DNA by reverse transcriptase and the resulting DNA was cloned into appropriate vectors. When introduced into cells, the DNA was copied into RNA that was infectious.142,168

Animal Viruses and Oncology Much of our present understanding of the origins of human cancers is a consequence of work on two major groups of animal viruses, retroviruses, and DNA tumor viruses (see Chapter 11). The first oncogene discovered was from the genome of Rous sarcoma virus. Remarkably, the virus had picked up a host gene that was involved in growth control.159 Since those seminal studies, virologists have identified a wide variety of oncogenes that have been captured by retroviruses (see Chapter 8). Additional cellular oncogenes were identified when they were activated by insertion of retroviral proviral DNA in or near the gene.75 When activated, these oncogenes are dominant and activate cell growth. The second group of genes that contribute to the origins of human cancers is the tumor suppressor genes that stop cell growth.109 Two such genes (p53 and pRB) are intimately associated with the so-called DNA tumor viruses that must replicate their DNA in cells that are not replicating. Genetic alterations at the p53 locus are the single most common mutations known to occur in human cancers—they are found in 50%-80% of all cancers.111 The p53 protein was first discovered in association with the SV40 large T-antigen, a viral gene essential for viral DNA replication.107,112 SV40, the human adenoviruses, and the human papillomaviruses all encode proteins that interact with and inactivate the functions of the retinoblastoma susceptibility gene product (Rb) and p53.39,43,107,112,150,184,185 Our understanding of the roles of cellular oncogenes and the tumor suppressor genes in human cancers would be far less significant without the insight provided by studies with these viruses.

Viruses associated with cancers have provided some of the most extraordinary episodes in modern animal virology.126 The recognition of a new disease and the unique geographic distribution of Burkitt lymphomas in Africa17 (see Chapter 11) set off a search for viral agents that cause cancers in humans. From D. Burkitt24 to Epstein, Achong, and Barr56 to W. Henle and G. Henle,78 the story of the Epstein-Barr virus and its role in several cancers, as well as in infectious mononucleosis, is a science detective story without rival. Similarly, the identification of adult T-cell leukemia, a new pathologic disease, in Japan by K. Takatsuki176 led to the isolation of human T-cell leukemia virus (HTLV-1) by I. Miyoshi and Y. Hinuma.138 It was soon realized that this virus had been identified previously by Gallo and his colleagues. Even with the virus in hand, there is still no satisfactory explanation of how this virus contributes to adult T-cell leukemia.

An equally interesting detective story concerns hepatocellular carcinoma and the discovery of hepatitis B virus. By 1967, S. Krugman and his colleagues103 had strong evidence indicating the existence of distinct hepatitis A and B viruses, and in the same year B. Blumberg20 had identified an unusual protein called the Australia antigen. Through a tortuous path, it eventually became clear that the Australia antigen was the coat protein of hepatitis B virus. Its detection was a predictable indictor for the presence of hepatitis B virus. Although this discovery ultimately freed the blood supply of this dangerous virus, Hilleman at Merck, Sharp, and Dohme and the Chiron Corporation (which later isolated the hepatitis C virus) went on to produce the first human vaccine that prevents hepatitis B infections and very likely hepatocellular carcinomas associated with chronic virus infections (see Chapter 15). The idea that a viral vaccine could prevent cancer (first proven with the Marek disease virus and T-cell lymphomas in chickens)18,47 came some 82-85 years after the first discoveries of tumor viruses by Ellerman, Bang, and Rous.

Vaccines and Antivirals Among the most remarkable achievements of the 20th century is the eradication of smallpox, a disease with a >2000-year-old history.76 In 1966, the World Health Organization began a program to immunize all individuals who had encountered an infected person. This strategy was adopted because it simply was not possible to immunize entire populations. In October 1977, Ali Maolin of Somalia was the last person in the world to have a naturally occurring case of smallpox (barring laboratory accidents). Because smallpox has no animal reservoir and requires person-to-person contact for its spread, most scientists agree that we are free of this disease as a natural infection.76 Consequently, most populations have not maintained immunity to the virus and the world’s populations are becoming susceptible to infection. Many governments now fear the use of smallpox virus as a weapon of bioterrorism, and the debate continues over whether to destroy the two known stocks of smallpox virus in the United States and Russia.77 As a consequence, the development of new, more effective vaccines and safe anti–smallpox virus drugs has risen high on the list of priorities for some countries and have already been stockpiled in the United States. It is paradoxical that humankind’s most triumphant medical accomplishment is now tarnished by the specter of biowarfare.

The Salk and Sabin poliovirus vaccines were the first products to benefit from the cell culture revolution. In the early 1950s in the United States, just before the introduction of the Salk vaccine, about 21,000 cases of poliomyelitis were reported annually. Today, thanks to aggressive immunization programs, polio has been eradicated from most of the world (see Chapter 11).132 However, complete eradication has proven to be difficult in some countries due to economic and political issues. With the substantial financial support of the Gates Foundation, there is hope that global immunization campaigns can lead to eradication of poliomyelitis from the planet.

The first viral vaccines deployed included infectious but attenuated viruses, inactivated virus preparations, and subunit vaccines comprising viral proteins and no viral nucleic acid. Both the Salk inactivated virus vaccine and the recombinant hepatitis B virus subunit vaccine were products of the modern era of virology. Today many new vaccine technologies are either in use or are being tested for future deployment.3,23,153 11

These include recombinant subunit vaccines, viruslike particle vaccines, viral antigens delivered in viral vectors comprising vaccinia virus or adenovirus, DNA plasmids that express viral proteins from strong promoters, and mRNA vaccines. Therapeutic vaccines boost the immune system using specific cytokines or hormones in combination with new adjuvants to stimulate immunity at specific locations in the host or to tailor the production of immune effector cells and antibodies. The first vaccines for smallpox were reported in the Chinese literature of the 10th century,57 but up until a few years ago, progress and new approaches had been slow.

Although vaccines have been extraordinarily successful in preventing specific diseases, up until the 1960s, few natural products or chemotherapeutic agents that cured or reduced viral infections were known. That situation changed dramatically with the development of Symmetrel (amantadine) by Dupont in the 1960s as a specific influenza A virus drug. Soon after, acyclovir, an inhibitor of herpesviruses, was developed by Burroughs-Wellcome. Acylovir achieves its remarkable specificity because to be active, it must be phosphorylated by the viral enzyme thymidine kinase before it can be incorporated into viral DNA by the viral DNA polymerase. This drug blocks HSV-1 and HSV-2 replication after reactivation from latency and stopped a growing epidemic in the 1970s and 1980s (Chapter 14). The development of other nucleoside analogs that inhibit viral replication has led to many compounds effective against DNA viruses. Until the HIV epidemic, few drugs effective against RNA viruses other than the influenza A virus were known. That issue has changed dramatically in the past 10 years as many new antivirals effective against different RNA viruses, particularly HIV and HCV, have been discovered. As natural products, the interferons are used successfully in the clinic for hepatitis B and C infections, cancer therapy, and multiple sclerosis. The interferons, novel cytokines, produced by infected cells were found while studying virus interference.23,88,89 They modulate the immune response and continue to play an increasing role in the treatment of many clinical syndromes.

Virology and the Birth of Immunology Edward Jenner was a British surgeon who is credited with making the first smallpox vaccine in 1796 and has also been called the “father of immunology.” Jenner began a long tradition of virology providing seminal discoveries about the immune response. Two examples will serve to illustrate this pattern. Alick Issacs and Jean Lindenmann, while working at the National Institute for Medical Research in London, found that addition of heatinactivated influenza virus to the chorioallantoic membrane of chicken eggs interfered with the replication of influenza virus. When they published this observation in 1957, they coined the term “interferon”.88 In the 1970s, the protein was purified from cells by Sidney Pestka and Alan Waldman,147 and subsequently the genes encoding the proteins were cloned.71 This achievement allowed formal proof that IFN—by that time known to comprise a variety of different proteins—could interfere with viral replication. Extensive work with viruses showed that IFNs bind to cell surface receptors, and through the JAK-STAT signal transduction pathway, induce the synthesis of over 1000 mRNAs whose products establish an antiviral state in that cell.38 Interferons protect against both viral and bacterial infections and also play a role in tumor clearance.

While working at the John Curtin School of Medical Research in Australia, Rolf Zinkernagel and Peter Doherty provided seminal insight on how cytotoxic T cells (CTLs) recognize virus-infected cells. They were studying mice infected with lymphocytic choriomeningitis virus (LCMV). Because this virus is noncytopathic, they hypothesized that brain damage in infected mice was a consequence of CTLs attacking virus-infected cells. They saw that CTLs isolated from LCMV-infected mice lysed virus-infected target cells in vitro only if both cell types had the same MHC haplotype. This requirement was termed MHC restriction.194 In other words, a CTL must recognize two components on a virus-infected cell, one virus-specific, and one from the host. Subsequent research revealed that CTLs recognize a short viral peptide bound to MHC class I proteins on the surface of target cells. These observations revolutionized our understanding of T-cell mediated killing, establishing a foundation for understanding the general mechanisms used by the immune system to recognize both foreign microorganisms and self-molecules. The results have had wide implications for clinical medicine, not only in infection but also in areas such as cancer and autoimmune reactions in inflammatory diseases.

Emerging Viruses In general, emerging viruses cause human infections that have not been seen or reported before (see Volume 1). They usually attract the public’s attention, often by media sound bites like “killer viruses emerge from the jungle.” The fact is that spread of infections through different hosts is well known in virology. Most so-called emerging infections represent zoonotic infections: infection of humans by a virus that normally exists in an animal population in nature.172 Typically, these viruses have RNA genomes and some are spread to humans by insect vectors.

Perhaps the most infamous emerging virus infection of the 20th century is the human immunodeficiency virus, HIV-1, a retrovirus.82 Progenitor HIV viruses exist in primates and we now believe they infected humans as a result of hunting and slaughter for food.155 HIV was first recognized as a new disease entity by clinicians and epidemiologists in the early 1980s, and they rapidly tracked down the venereal mode of virus transmission. The virus was detected in blood products and transplant tissue. The immune system of HIV-infected individuals is severely compromised, which results in a variety of infections by usually benign microbes. The first published report of acquired immunodeficiency syndrome (AIDS) was in June 1981. Possible causative agents were first suggested in 19837 and then 1984.66 Had this pandemic occurred in 1961 instead of 1981, neither the nature of retroviruses nor the existence of its host cell (CD-4 helper T-cell) would have been understood. HIV is a lentivirus (lenti is Latin for slow), and despite its recent appearance in humans, lentiviruses have been around for a long time. In fact, one of the first animal viruses to be identified in 1904 was the lentivirus that causes infectious equine anemia.

Many other examples of emerging viruses have attracted global concern and an exceptional rapid response of scientists and health officials.172 The SARS and West Nile virus epidemics revealed the presence of a new human corona virus (SARS-CoV 1), identified with unprecedented speed, and the invasion of an Old World virus into the western hemisphere (West Nile virus).86,131 In 2006, Chikungunya virus (an endemic virus infection in Africa) spread explosively to several countries where it was hitherto unknown.154 On Reunion Island, more than 40% of the population of 800,000 people was infected. Other emerging infectious agents include Ebola virus, Lassa fever virus, Zika virus, and many mosquito-borne viruses. The first appearance of avian H5N1 virus in humans in 1999 produced fears of a pandemic of serious proportions since humans had no immunological history of infection by this avian strain.167 Soon thereafter, the emergence of the pandemic H1N1 influenza virus in 2009 produced similar worries because of the relationship of the virus to the deadly 1918 influenza epidemic.169 The mobilization of world health networks, public health officials, vaccine producers, veterinarians, clinicians, and molecular virologists marked a new chapter in dealing with emerging diseases. As the COVID-19 pandemic revealed, emerging viruses are and will be ongoing features of viral infections. There will 12

always be another potential pandemic in our future.

Epidemiology of Viral Infections The study of the incidence, distribution, and control of disease in a population is an integral part of virology. The technology advancements of the last 50 years have provided epidemiology with a terrific boost. The discovery of specific molecular reagents (eg, recombinant DNA technology, antibodies, polymerase chain reaction [PCR], rapid diagnostic tests, high volume DNA and RNA sequencing) now enables detection of virions, proteins, and nucleic acids in body fluids, tissue samples, or in the environment. Moreover, we now can compare and classify viral isolates rapidly, determine the relationships between virus strains, and track the spread of infections around the world. The marriage of behavioral, geographic, and molecular epidemiology made this a most powerful science.48

The understanding of epidemics and pandemics of our most common viral infections such as influenza requires the perspective of ecology, population biology, and molecular biology.100,167,169 G. Hirst and his colleagues (1941–1950) developed the diagnostic tools that permitted both the typing of the hemagglutinin (HA protein) of influenza A strains and the monitoring of the antibody response to this antigen in patients (see Chapters 42 and 43). These observations have been expanded, with more and more sophisticated molecular approaches, to prove the existence of animal reservoirs for influenza viruses, the reassortment of viral genome segments between human and animal virus strains (antigenic shift), and a high rate of mutation (antigenic drift) caused by RNA-dependent RNA synthesis with no known RNA editing or corrective mechanisms.143 These molecular events that lead to episodic local epidemics and worldwide pandemics are understood in broad outline. Many viruses such as SARS CoV-2 are now known to evolve at high rates following basic darwinian principles in a time frame shorter than that of any other organism. Indeed, we now understand that RNA virus populations exist as a quasispecies or a swarm of individual viral genomes where every member is unique. Influenza viruses are successful because they have evolved to carry the very engines of evolution: mechanisms of mutation and recombination (reassortment). Influenza A virus has not been eliminated even with effective vaccines and antiviral drugs. Variants always arise that escape effective immune responses thorough high mutation (drift) and when coinfection occurs with viruses spreading from nonhuman hosts, new reassortants regularly arise. These new combinations of viral genes can change the pattern of infection from local to pandemic via an antigenic shift of its HA and NA subunit genes. These studies reveal an extraordinary lifestyle that reverberates around the planet in birds, farm animals, and humans. The study of the mechanisms of viral pathogenesis and modulation of the immune system has led to new insights in the virus-host relationship.

New technology discovered and developed over the past 35 years is changing the way viral infections are studied in the lab and in the field and is changing our appreciation of epidemiology and virus ecology.166 Amplification technologies such as PCR permit rapid sampling of viral nucleic acids without growth in culture or plaque purification. Microarray technology where discriminatory DNA sequences from all sequenced viral genomes are put on a single array enables rapid classification of PCR amplified nucleic acids.179 Rapid genome sequencing has revealed hitherto described viral genomes, relationships among viruses, and sequence heterogeneity within a virus population.114 Mutations can be detected rapidly, documented, and localized in the viral genome. Importantly, the biological consequences can be monitored quickly. For example, in the late 1970s, viral epidemiologists were confronted with a highly transmissible, lethal infection of puppies.134 In record time, scientists found that just two mutations in the capsid gene of feline parvovirus altered the host range such that the mutant could infect dogs. In less than a year, a completely new, highly pathogenic virus called canine parvovirus spread all around the world. Its evolution has continued to be monitored and a highly effective vaccine was developed. A similar type of molecular archeology enabled scientists to analyze serum samples collected from patients in the 1950s in efforts to understand the origins of HIV.82 Sequence analysis of the HIV genome from one sample (ZR 1959) suggested that the virus may have emerged in the 1940s to 1950s. Field studies in Africa of viruses present in primate feces indicated that HIV most likely derived from a chimpanzee lentivirus in Africa.155 After the initial human infection, rapid mutation and selection established the first human variants of this lentivirus that replicated and continued to evolve as they spread through their new human hosts.

We cannot forget the considerable impact of veterinary virus epidemiology on our understanding of complicated human diseases. For example, careful epidemiological work by Sigurdsson and colleagues on unusual diseases of sheep160 provided the first understanding of slow infections in sheep (Visna-Maedi virus; a lentivirus) and infectious proteins (prions), which cause spongiform encephalopathies (Chapter 22).

As we will describe in the next section, molecular epidemiology is reaching new levels of sophistication, not only in detecting new viruses but also taking inventory of the viral ecosystem (the virome). Whether the next human epidemic will result from a novel variant of Ebola virus, coronavirus, or Norwalk virus or the more likely possibility of a new pandemic variant of influenza virus remains to be seen. The new technologies also enable analysis of virus populations in natural communities of nonhuman animals.143 What is abundantly clear, however, is that the demographics of the human population on earth are changing at unprecedented rates (see Table 1.2). Even as birth rates slow, our planet will house 8-10 billion people by 2050–2100. For the first time, there will be three to four times more people above the age of 60 than below 3-4 years of age. Not only are we an aging population but we also are moving to urban environments with more than 20-30 cities containing more than 10 million people. Clearly, patterns of human behavior (increased population density, increased travel, increased ages of the population, income disparity, food shortages, and climate change) will provide the environment for the selection of emerging viruses and the challenges to the new field of molecular epidemiology.

TABLE 1.2

Advances and Challenges

13

Host-Virus Interactions and Viral Pathogenesis The technologies that contributed most to the modern era of virology (1960 to present) were advances in cell culture and molecular biology.54 Virologists described the replicative cycles of viruses in great detail under well-defined conditions and demonstrated the elaborate interactions between viral genomes, viral proteins, and the host cellular machinery. As indicated previously, these advances resulted in an extraordinary inquiry into the functions of infected or uninfected host cells using the tools of both molecular and cell biology. As this approach matured, it became more reductionist in nature, and the questions became more detailed. However, some virologists used the new knowledge to move back to more complicated in vivo systems to study previously difficult problems in host-virus interactions involving the natural host or animal models of infection. Chief among these new questions was, how does a virus cause disease processes in the animal? How do we quantitate viral virulence and what is the genetic basis of an attenuated virus? How is a viral infection transmitted? These studies have identified, in selected viruses, a set of genes and functions that broadly impact our understanding of pathogenesis.

The COVID-19 Pandemic 14

(Section adapted from Ref.40)

The world-wide spread of SARS-CoV-2 was devastating on a scale that few could have imagined. While there have been multiple viral pandemics in the last century, two stand out for their impact: the 1918 influenza pandemic and acquired immune deficiency syndrome (AIDS). Both killed millions of people and have had broad social and economic impacts. At this writing in January 2022, there are more than 400 million documented cases of COVID-19 worldwide and over 6 million deaths. Given the difficulties in testing massive numbers of people, the true prevalence of infection and death is undoubtedly much higher. The explosive spread of SARS-CoV-2 in late 2019 and early 2020 caught most of the world unprepared in many ways. Initially, there were few ways to control the spread except by old-fashioned quarantine methods: most of the world’s population locked down, suspended travel, closed borders, and limited or stopped interactions of large groups of people. As a result, the medical disaster was compounded with social disruption and economic devastation. The COVID-19 causative agent was isolated rapidly, identified as a coronavirus, and named SARS-CoV-2. While a new member of the Coronaviridae, it was not a “new” virus because coronaviruses are well known. Indeed, the first human coronavirus was described in 1964 by June Almeida in London. There are at least four known human coronaviruses that cause mild respiratory infections, including the common cold. Other animals can be infected with particular coronaviruses such as mouse hepatitis virus, infectious bronchitis virus of chickens, and transmissible gastroenteritis virus of swine. SARS-CoV-2 joins severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses, which emerged in 2002 and 2012, respectively, that cause severe human disease. SARS and MERS share some features with COVID-19 but each also has unique aspects. The SARS-CoV epidemic ended after ~8000 documented infections and that virus has not been since isolated from humans. MERS-CoV continues to infect several humans each month, mainly in the Arabian Peninsula. Like most emerging viruses, SARS-CoV-2 most likely existed in an animal reservoir before it entered the human population. When and where SARS-CoV-2 entered humans has not been determined, although all evidence points to a bat origin: multiple SARS-CoV-2–related viruses have been identified from bats in China, Japan, Thailand, and Laos. The genome sequence of some of the first virus isolates in China was widely disseminated to the research community. Within a few weeks, a global army of investigators was mobilized to study this new agent. This intense, scientific effort led to the rapid characterization of the SARSCoV-2 attachment/fusion protein and the cell surface receptor was identified as the human enzyme ACE2, the same receptor used by SARS-CoV, the cause of SARS. Remarkably, the structure of the viral attachment protein (also called the spike protein) was determined at atomic-level resolution within a few months. Even more astounding was the development of new vaccines for production of the spike protein: mRNA vaccines, adenovirus vectored vaccines, and protein vaccines. Testing these vaccines for safety and efficacy was achieved faster than any other vaccine. The efficacy of these vaccines is nothing less than amazing. Nevertheless, the challenges of manufacturing, distributing, and public acceptance have been substantial.

Lessons Learned (Section adapted from Ref.40)

The most important lesson is that future pandemics are inevitable, and we must be ready for them. If we learned anything by being caught by surprise by the explosive spread of SARS-CoV-2, it is that rapid methods of surveillance and data sharing are essential. One challenge is how to recognize potential pandemics early and inform the public to allow prompt public health interventions. The world must have coordinated plans for action, to avoid confusion and inadequate responses. A second challenge is that we must identify and stockpile antiviral compounds and vaccines that can inhibit replication by a wide range of coronaviruses. Such therapeutics must have been tested for safety in humans so that at the first signs of a new outbreak they could be rapidly deployed. We learned that transmission of infection can be slowed by physical distancing, wearing masks, washing hands, and staying home when ill. These efforts are classical public health procedures that we must not forget. An important lesson is that research in basic virology, cell biology, immunology, biochemistry, and other relevant areas is essential. These studies provide the necessary head start to meet new viruses before they overtake us and our health system, economy, and society. The fruits of such research enabled the rapid development of diagnostic tests, the use of DNA sequencing methods to characterize millions of genomes, and of course novel vaccines that are changing the face of the pandemic. While these lessons are obvious, a more difficult challenge has emerged. COVID-19 has revealed deficiencies in how different societies operate and communicate as well as how politics can color public health responses. Since we live in a world of instant communication, dissemination of information, without regard to accuracy, is fast and seemingly uncontrollable. Moreover, the unwillingness of some institutional and political leaders as well as some members of the public to follow science-based guidelines and protocols is a serious problem that will continue long after the COVID-19 pandemic eases. More education is essential because much of the public and many of our public officials barely had any concept of viruses as infectious agents and their potential to upend our world. Until the public has some basic understanding of virology, history will repeat itself.

Past is Prolog (Section adapted from Ref.55)

The COVID-19 pandemic not only changed the world but also changed the study of virology as well. In general, virology has always been shaped by technology. When a virologist wants to answer a question, and current methods are inadequate, the virologist invents, borrows, or repurposes to develop a new technique. Many techniques and methods deployed to study SARS-CoV-2 and COVID-19 were not available just a few years ago. Moreover, the integration of new technology with public health measures to detect viral genomes and proteins as well as the immune response to infections has been revolutionary. New vaccine technology has enabled discovery, production, and mobilization of vaccines with remarkable efficacy. Deep, high-throughput sequencing, the relatively unbiased methods to determine the entire genome sequence of viruses both known and unknown, has revolutionized virology because it enables characterization of millions of genomes in a short time. Molecular epidemiology, learning about the transmission of infections in real time, and the study of virus evolution have been changed forever by this new technology. At the molecular level, structural biology has been changed dramatically by cryoelectron microscopy. The atomic structure of individual viral proteins or intact virus particles, which used to take many years dominated by trial and error, can be determined in less than a month. Artificial 15

intelligence programs have revolutionized the prediction of protein structures. Antiviral drug discoveries that relied on painstaking work to identify potential molecular targets followed by tedious screens are relics of the past. With new genetics technology, imaging, and powerful chemistry methods, potential cellular targets for antivirals can be identified in weeks. The COVID-19 pandemic mobilized powerful technology leading to rapid applied and fundamental progress. The drive to develop therapeutics and vaccines as well as to discover how viruses work was remarkable and productive. The testing and Food and Drug Administration emergency use authorization of two vaccines within less than a year of the first genome sequence being posted—and more remarkably, both based on a vaccine platform that had not been previously used at any scale in humans for vaccine purposes—is without precedent. The kinetics and scale of molecular epidemiology is remarkable. With millions of SARS-CoV-2 genome sequences, we know almost as much about the evolution of this virus as any other. Fortunately, the pace of work and depth of analysis are applicable to many viruses, not just coronaviruses or the next pandemic virus. These technological advances move all of biology forward. In the midst of this worldwide calamity, the new revolution in virology brings hope for the future. There will be another pandemic, and lessons learned from this one will help us with the next.

The Future of Virology The future of virology is unpredictable but is guaranteed to be exciting. Who knows what discoveries remain? Certainly, the number of astounding and groundbreaking developments in biology over the past 60 years is remarkable.54 Most could not have been predicted or even imagined, prior to their discovery. That virologists participated in making many of these discoveries is no accident: viral gene products have evolved to engage all the key nodes of biology ranging from the atomic to the organismal. We only must be smart enough to figure out how to identify these nodes. The forces that will drive our field are technology development, public health, information processing, and, of course, personal curiosity. Indeed, new life science technologies invariably will give rise to new, unexpected insights in virology to meet our current challenges. That has been and continues to be the future of virology (see Table 1.2).

A Role for Systems Biology in Virology Not too long ago, molecular virology was limited to studies of one virus and one gene or gene product at a time. More complex studies often were seen as “descriptive.” Times have changed! New technology enables virologists to interrogate simultaneously many viruses and large groups of genes or gene products in ever expanding environments and biological networks. In this context, a network is defined as the interconnected intracellular processes that control everything within a cell: for example, DNA replication, processes of gene expression, organelle biogenesis, and metabolism to name a few.130 The definition also encompasses networks of intercellular communication at the tissue, organ, and wholeorganism level. Virologists are beginning to embrace a tenet of systems biology where information flows through these networks and disease arises when these networks are perturbed. Viral gene products cause changes in network architecture and thereby alter the dynamics of information flow. Future studies of viral pathogenesis are likely to involve identification and understanding of specific viral signatures of network imbalance that do not affect just one pathway but alter the fundamental homeostatic balance.19,54,141,164

Genomics and the Predictive Power of Sequence Analysis The development of technological advances in biology often drives new approaches and permits one to ask novel questions that could not even be framed in the past. In the last decade of the 20th century, rapid and inexpensive DNA sequencing methods opened the way to sequence the genomes of many viruses and their hosts. This created large databases containing information about the variation of DNA or RNA sequences within a single virus (eg, HIV, influenza, coronaviruses) and permitted predictions about the nature of the mutations driving selective changes, mutation frequencies of different viruses, and evolutionary changes from isolates around the world. The correlations of these sequence variations with drug and vaccine resistance, changes in the genetic background of the host, and virulence have been informative. Host genomes contain an amazing number of viral or viral-related sequences.11,12 More than 50% of the DNA sequences found in the human genome were derived from retroviruses, retrotransposons, DNA transposons and randomly amplified sequences of genes (SINES and the 7S RNA gene), pseudogenes, and repetitive DNA sequences.106,177 Viruses certainly have left a major mark upon the evolution of their host’s genomes in addition to the selective pressures they exert via virus infections and deaths. During the evolution of humans from their ancestral line, retroviruses and retrotransposons (the LINE-1 elements) have entered the germ line, amplified their copy numbers, and integrated at various sites in the genome. This process introduces mutations, alters patterns of gene expression, and creates new interactions of viruses with their hosts. This is clearly one of the drivers of host evolution. Over time, these retroviruses (human endogenous retroviruses or HERVs) accumulate mutations in their genes, and some recombine out of the genome leaving only the LTRs as a remnant marking their past insertion. While humans no longer contain viable HERVs, the multiple copies of HERV-H or HERV-K viruses when transcribed in cells produce functional viral proteins from different copies of these viruses, and the viral particles that are produced are defective and very poorly transmitted. Cellular transcription factors regulate the expression of the HERVs, and the p53 transcription factor, activated by stress and DNA damage, transcribes the HERV H genome and produces particles in response to such stress.192 Similarly, the LINE-1 retrotransposons, which have about 300 viable and movable elements in the human genome today, are responsible for about 1% of the mutations found in each generation. Line-1 transposons also contain p53 DNA response elements83 and are also regulated by stress responses recorded by the host. While it is clear that retroviruses and transposons can shape the host genome, it is equally clear that the host genome is a place for new viral genomes to evolve, recombine with exogenous viral genomes and possibly produce a new agent optimized for replication in its host. Understanding the dynamics of these vestiges of viruses that reside in our genome is a challenge for the future.

With many host genome sequences representing all kingdoms of life in the databases, it has been possible to do some rather eye-opening analyses. For example, the resurrection of endogenous retroviruses from inactive sequences in host DNA has allowed the investigation of interactions between extinct pathogens called paleoviruses and their hosts that occurred millions of years ago.52 By cloning these sequences, it has been possible to identify the cellular receptor of these extinct retroviruses.157 Perhaps more amazing is that similar “viral genome fossils” representing DNA copies of filoviruses and bornaviruses as well as parvoviruses and circoviruses have been found in a variety of host genomes.11,12 When the evolutionary history of various host genomes harboring these viral sequences were compared, it was possible to deduce that ancestors of modern viruses were in existence millions of years ago. What is even more curious is that these genome insertion events seemed to happen around the same time in a wide variety of mammals. What global event could have stimulated such activities? 16

The Virome: How Many Viruses Are There and Where Are They? Virus ecology, as a result of modern virus discovery technology, is posing many questions.100,166 In 1977, when Fred Sanger sequenced the DNA genome of coliphage phiX174, many virologists were impressed with the wealth of information contained in a “simple” DNA sequence and the congruence of genetic and biochemical data with the genome structure. The recent discovery of “giant viruses” that infect amoeba revealed that the time-honored definition of viruses as small filterable agents no longer holds (Chapter 17).

In 1 infectious unit/cell, will accumulate spontaneously deleted defective particles that are maintained during passage by the presence of complementing wild-type helper virus.168 Passage of the same virus at very low MOI (eg, 0.01 infectious units/cell) discourages the accumulation of defective particles because few cells will be coinfected with an infectious and a defective particle, and defective particles cannot replicate in the absence of a wildtype helper. Conversely, other experiments, such as metabolic labeling, assessing the host response to infection, or quantifying the activity of a virally encoded enzyme, are done at high MOI (eg, 10 infectious units/cell) to ensure that all cells in the culture are infected and that the infection is as synchronous as possible. For such experiments, use of too low an MOI may result in an apparently asynchronous infection and a high background owing to the presence of uninfected cells in the culture.

The Poisson distribution can be used to predict the fraction of cells in a population infected with a given number of particles at different multiplicities of infection. As applied to virus infections, the Poisson distribution can be written as:

where P(k) equals the probability that any cell is infected with k particles, m equals MOI, and k equals the number of particles in a given cell. Sample solutions to the equation are shown in Table 2.2 for commonly used multiplicities of infection.184 Inspection of this table and consideration of the error inherent in any virus titration involving a serial dilution leads to some significant practical guides in experimental design. Note first that in a culture infected at an MOI of 1 PFU/cell, 37% of cells remain uninfected—an unacceptably high number for an experiment designed to measure a single round of synchronous infection. An MOI of at least 3 is required to infect 95% of the cells in culture. A shortcut is to calculate the fraction uninfected cells by setting k = 0, which reduces the equation to e−m, from which the fraction of cells that are uninfected can be easily calculated. Given that titers can easily be inaccurate by a factor of two, the use of a calculated MOI of 10 ensures that 99% of the cells in a culture will be synchronously infected even if the measured titer is twofold higher than the actual titer.

TABLE 2.2

The Poisson distribution for multiplicity of infection (MOI)

36

One-Step Growth Experiment The one-step growth experiment is a classic assay developed initially for bacteriophage40 and still frequently used to determine the essential growth properties of a virus. The goal of this experiment is to measure the time course of virus replication and the yield of virus per cell during a single round of infection. The experiment is carried out as follows. Several dishes containing confluent monolayers of an appropriate cultured cell are infected simultaneously with virus at a high MOI (eg, 10 PFU/cell). After an adsorption period, monolayers are washed to remove unabsorbed virus and then incubated in culture medium. At various times after infection, virus from individual dishes is harvested, and at the completion of the experiment, the virus titer in samples representing each time point is determined. The virus yield at each point can be converted to PFU/cell (also called burst size) by dividing the total amount of virus present in the sample by the number of cells originally infected in the sample. Two examples of one-step growth experiments with coxsackievirus B3 or Rio Bravo virus are shown in Figure 2.8. Several features of the growth curve are noteworthy. First, during the first several hours of the infection, the titer in the cultures decreases and then increases. This initial dip in the growth curve is called eclipse and results from the fact that early during the experiment, virus attached to the cell surface but not uncoated remains infectious; however, infectivity is lost following uncoating during the first few hours of infection, and infectivity is recovered only after new virus is produced. The infection then enters a rapid growth phase, followed by a plateau. The plateau results from the fact that all infected cells have reached the maximum yield of virus, or have died or lysed, depending on the type of virus infection. The time interval from infection to plateau represents the time required for a single cycle of growth, and the yield of virus at plateau shows the amount of virus produced per cell. The experiment in Figure 2.8 suggests that coxsackievirus B3 has a relatively fast replication cycle, with progeny viruses detectable at between 5 and 7 hours postinfection. Conversely, Rio Bravo virus is not detected until 16 hours. One-step growth experiments to determine the length of a single replication cycle are ideally performed in the target cell of interest, as replication dynamics can differ between cell types, particularly depending on whether cell intrinsic antiviral pathways are intact.

FIGURE 2.8 One-step viral growth experiments. Left. Caco-2 cells were infected with coxsackievirus B3 at an MOI of 0.1, and progeny viruses were collected at a variety of time points followed by plating dilutions for plaque assays on HeLa cells. Data are presented as plaque forming units (PFU) per cell. (Courtesy of Dr. Yao Wang.) Middle. Similar to above, Huh7.5 cells were infected with Rio Bravo virus at an MOI of 5, and progeny viruses were titered on BHK cells. (Courtesy of Dr. Ian Boys.) Right. Human STAT1−/− fibroblasts were infected with VSV-GFP at an MOI of 5, and serially collected supernatants containing progeny viruses were used undiluted to reinfect naive cells. Intact cells were harvested, fixed with paraformaldehyde, and the percentage of infected (GFP+) cells was quantified by flow cytometry.

Multiplicity of infection is a critical factor in the design of a virus growth experiment. A true one-step growth experiment can only be done at high MOI. If the MOI is too low and a large fraction of cells are left uninfected, then virus produced during the first round of infection will replicate on previously uninfected cells, and thus multiple rounds of infection rather than one round will be measured. A growth experiment done at low MOI has utility in that it measures both growth and spread of a virus in culture; however, the time from infection to plateau does not accurately reflect the time required for a single cycle of infection. It is also noteworthy that some mutant phenotypes are multiplicity dependent.11

Modern Viral Assays and Approaches As with all fields of science, virology has modernized significantly over the past couple of decades. Whereas classical virology has often focused on the virus side of “virus-host interactions,” modern approaches are increasingly considering the host side, including a detailed examination of host factors required for viral replication, as well as host responses to viral infection, in particular the immune response. This chapter primarily emphasizes approaches to study virology that are more consistent with classical themes in the field, for example, understanding viral gene function, viral replication kinetics, and virus evolution. However, we also briefly discuss modern approaches that are being used to complement classical virology on the virus side and to augment our understanding of virus-host interactions from the perspective of the host.

Viruses Expressing Foreign Reporter Genes Genetic approaches have allowed many viruses to be modified to express a variety of reporter genes from the viral genome. Historically, these recombinant viruses expressed foreign genes such as chloramphenicol acetyltransferase or β-galactosidase, which could be monitored by enzyme assay and used as surrogates for viral gene expression. These strategies have largely been supplanted by fluorescent or bioluminescent reporters such as green fluorescent protein (GFP) and firefly luciferase, respectively. Reporter viruses have distinct advantages and limitations relative to their nonreporter parental viruses. Viruses expressing fluorescent reporters such as GFP can be used in cell culture to quantify viral infectivity, that is, the percentage of cells that become infected. Infectivity is typically quantified by flow cytometry or fluorescence microscopy and can then be used to calculate viral titers based on Poisson distribution. Titers can be expressed as “fluorescent cell units,” which is analogous to the plaque-forming units derived from plaque assays. However, the timing of viral replication needs to be considered and restricted to the first replication cycle, as viral spread will lead to more GFP-positive cells and an inaccurate assessment of true infectivity. Infectivity, as reflected by GFP positivity, can be used in many other applications. For example, a onestep growth experiment can be performed as described above, with the difference being that progeny viruses are detected by re-infecting naive cells and assessing infectivity by flow cytometry. An example is shown in Figure 2.8 with VSV-GFP. These data indicate that a single replication 37

cycle is between 5 and 6 hours. In this experiment, supernatants containing progeny viruses were not diluted as is standard in a plaque assay. Thus, this type of one-step growth curve can only determine the timing of a replication cycle, but it does not give information on how many infectious units of virus are produced per cell. This latter metric could be obtained if supernatants were serially diluted, and the data were backcalculated based on the Poisson distribution. Infectivity can simultaneously be complemented with quantitation of GFP mean fluorescence intensity (MFI). Depending on the virus, GFP MFI can provide distinct information about the viral infection process. For example, many GFP-expressing RNA viruses express the reporter from the viral genome similar to other viral proteins. Thus, GFP serves as a surrogate for viral protein synthesis, and the effect of any perturbation (eg, drug treatment, genetic modification, etc.) can be quantified by GFP MFI. This also holds for viruses expressing luciferase; indeed, many drug screening efforts now rely on viral genomes expressing luciferase for high-throughput assays (discussed below). For DNA viruses with highly defined temporal gene expression patterns, such as herpesviruses or adenoviruses, GFP can inserted into specific regions of the genome and used as a surrogate to monitor the temporal dynamics of viral gene expression. In other genetic strategies, GFP can be fused to a viral protein, and provided that the fusion does not disrupt viral protein function, the expression, subcellular localization, and dynamics of the viral protein can be monitored with a variety of microscopic techniques. Both fluorescent and bioluminescent reporters can additionally be used to monitor viral dynamics in vivo. For example, a virus expressing firefly luciferase can be administered to a mouse, and after a defined amount of time, the animal can be injected with luciferin substrate and imaged to assess which organs and tissues are productively infected (Fig. 2.9).

FIGURE 2.9 In vivo bioluminescence imaging. Mice were mock-infected (right) or infected with 106 PFU with gammaherpesvirus MHV68 expressing firefly luciferase under control of the M3 promoter (left). To quantitate luciferase activity, mice were injected intraperitoneally with luciferin and imaged within 20 minutes of injection. (Courtesy of Dr. Tiffany A. Reese.)

Reporter viruses have important limitations. Some viruses may not tolerate large foreign gene inserts due to packaging limits, negative effects on replication, or disruption of important secondary structural features in the viral genome. If the reporter gene is successfully inserted, the reporter virus is frequently attenuated relative to the parent virus. Additionally, the reporter gene may not remain stable over time. This is particularly true with +ssRNA viruses expressing foreign genes. After serially passaging, and in some cases after even a single passage, the foreign insert is lost, likely due to a recombination event that reverts the virus to “wild type.” Thus, generating stocks from passaged virus is usually a suboptimal approach. Summarily, while reporter viruses are powerful tools, they have several limitations, which must be considered during experimental design.

High-Throughput Approaches to Examine Virus-Host Interactions The field of virology has been highly amenable to advances in high-throughput approaches. Key aspects of high-throughput approaches include the ability to assay hundreds, thousands, or even tens of thousands of samples in a relatively short time frame. This is accomplished by sophisticated instrumentation such as luminometers, flow cytometers, or high-content microscopes, each of which can handle multiwell plates (eg, 96-, 384-, or 1536-well plates). Early examples of high-throughput approaches in virology include screening of a 100 000-compound library for inhibitors of SARS-CoV using cell viability as the readout.145 Many high-throughput approaches are limited, because they can only provide readouts at a single time point. For example, a drug screen that quantifies protection from virus-induced cell killing will typically be performed at a single time point that has been predetermined using known controls. Similarly, a high-throughput screen for antibodies that inhibit infection by a GFP-expressing virus will usually use a single infection time point for the final readout. However, newer live cell imaging and analysis platforms (eg, Incucyte) allow for real-time quantitative live cell imaging, thus providing finely resolved temporal resolution of viral replication and/or virus-induced changes to the host cell.

Rapid technological advances and decreasing costs of nucleic acid synthesis have revolutionized high-throughput genetic screens across all areas of biology, including virology. After the discovery of RNA interference in Caenorhabditis elegans, gene silencing technology based on small interfering RNA (siRNA) quickly became the method of choice for silencing genes in mammalian cells. Targeted and genome-scale siRNA libraries were developed and screened in cell-based assays to identify host factors that were required for viral replication. The first genome-scale screens were performed with HIV-1,9,92,183 with many other viruses to follow. These screens provided a wealth of new information about the specific host proteins required for infection and, in some cases, host factors that contributed to cell intrinsic control of viral replication. However, screening hits from lab to lab did not have extensive overlap,10 raising concerns about the utility of this method. Similar to RNAi/siRNA technology, the CRISPR antiviral pathway in prokaryotes has also been co-opted to modulate gene expression in eukaryotic cells. CRISPR-based strategies can be used to (1) knock genes out by targeting DNA at the gene of interest, (2) reduce gene expression by targeting mRNA, or (3) induce gene expression by promoting gene-specific transcription. To date, the field of virology has gained the most traction with genome-scale CRISPR knockout libraries, which have been used to identify with high confidence and reproducibility host factors required for multiple viruses, including flaviviruses,104 HIV-1,122 influenza A virus,59 and alphaviruses.182 Conversely, several genome-scale CRISPR-based screens have also been used to identify inhibitory host factors targeting viruses such as influenza A virus, yellow fever virus, and murine norovirus.66,120,135 Given the low cost and relative ease of performing CRISPR screens, this technology is certain to continue revealing host genes with prominent roles during virus-host interactions. 38

Virus Genetics Viruses are subject to the same genetic principles at work in other living systems, namely mutation, selection, complementation, and recombination. Genetics impacts all aspects of virology, including the natural evolution of viruses, clinical management of infections, and experimental virology. For example, antigenic variation, which is a direct result of mutation and selection, plays a prominent role in the epidemiology of influenza virus and HIV in the human population, and mutation to drug resistance offers a significant challenge to the clinical management of virus infections with antiviral drugs. This section deals primarily with the application of experimental genetic techniques to basic virology. A major goal of experimental virology is to understand the functional organization of a viral genome. This means determination of the structure of a virus genome at the nucleotide sequence level, coupled with isolation and characterization of mutant viruses. Thus, genetic analysis of viruses is of fundamental importance to experimental virology. Before the advent of modern nucleic acid technology—that is, during a classical period of forward genetics—genetic analysis of viruses consisted of the random, brute force isolation of large numbers of individual virus mutants, followed first by complementation analysis to determine groupings of individual mutants into genes, then recombination analysis to determine the physical order of genes on the virus genome, and finally the phenotypic analysis of mutants to determine gene function. This approach, pioneered in the 1940s through the 1960s in elegant studies of several bacteriophage, was the primary method for identifying, mapping, and characterizing virus genes. The application of cell culture techniques to animal virology opened the door to classical genetic analysis of animal viruses, resulting in a flurry of activity in the 1950s through the 1970s, during which time hundreds of mutants were isolated and analyzed in prototypical members of most of the major animal virus families.51 Nucleic acid technology introduced in the 1970s brought with it a variety of techniques for physical mapping of genomes and mutants, including restriction enzyme mapping, marker rescue, and DNA sequence analysis, which together replaced recombination analysis as an analytic tool. Mutants and techniques from the classical period continue to be of enormous utility today; however, recombinant DNA technology has brought with it reverse genetics, in which the structure of the genome is determined first using entirely physical methods, then the function of individual genetic elements is determined by analyzing mutants constructed in a highly targeted fashion. Furthermore, modern low-cost sequencing technology now means that thousands of complete viral genomes can be generated daily. However, additional studies are still required to understand genome structure-function.

The genetic approach to experimental virology has the profound advantage of asking of the organism under study only the most basic question— What genes are needed to survive and why?—without imposing any further bias or assumptions on the system. Organisms often respond with surprises that the most ingenious biochemist or molecular biologist would never have imagined. What follows is a summary of the critical elements of both the classical and modern approaches to virus genetics as applied to experimental virology.

Fundamental Genetic Concepts Concepts fundamental to genetic analysis of other organisms apply to genetic analysis of viruses, and a clear understanding of these concepts is essential. The most important of these concepts are briefly summarized next.

Wild Type, Mutations, and Mutants Wild type refers to a genotype or phenotype that predominates in nature. It is important to understand that in the context of experimental virus genetics, a virus designated as wild type can differ significantly from the virus that actually occurs in nature. For example, virus genetics often relies heavily on growth and assay of viruses in cell culture, and natural isolates of viruses may undergo significant genetic change during adaptation to cell culture. In addition, viruses to be designated as wild type should be plaque purified before initiating a genetic study to ensure a unique genetic background for mutational analysis. Mutation refers to a sequence alteration in the nucleic acid genome of a virus, whereas viruses containing mutation(s) in their genomes are referred to as mutants.

Genotype and Phenotype Genotype refers to the actual genetic change from wild type in a particular virus mutant, whereas phenotype refers to the measurable manifestation of that change. This distinction is emphasized by the fact that a single genotype may express different phenotypes depending on the assay. Thus, for example, the same missense mutation in a virus gene may cause temperature sensitivity in one cell line but not another, or a deletion in another virus gene may have no effect on the replication of virus in culture but may alter virulence in an animal model.

Selection and Screen Selection and screen refer to two fundamentally different methods of identifying individual virus variants contained in a mixed population of viruses. Selection implies that a condition exists where only the desired virus will grow, and growth of unwanted viruses is suppressed. Thus, a drug-resistant virus can be identified by plating a mixture of wild-type, drug-sensitive, and mutant, drug-resistant viruses together on the same cell monolayer in the presence of the inhibitory drug, thereby selecting for drug-resistant viruses that grow and against wild-type viruses that do not grow. A screen implies that both the desired virus variant and one or several other unwanted virus types grow under a given condition, such that many viruses must be analyzed individually to identify the desired variant. For example, in searching for a temperature-sensitive mutant (ie, a virus whose growth is inhibited relative to wild-type virus at an elevated temperature), no condition exists under which the mutant alone will grow. Therefore, virus must be plated at a low temperature where both wild-type and mutant virus will grow, and plaques tested individually for temperature sensitivity. Sometimes a screen can be streamlined by introducing a phenotypic marker into the variant of choice. For example, a knockout virus might be constructed by inserting reporter genes such as β-galactosidase or GFP into the virus gene to be inactivated. Thus, viruses containing the insertional knockout can be distinguished from unmodified viruses.180 This latter example is still a screen, because both wild-type and mutant viruses grow under the conditions used; however, the screen is simplified because mutant viruses can be readily identified by their color, obviating the need to pick and test individual plaques. Selections have considerable advantages over screens but are not always possible.

Essential and Nonessential

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The terms essential and nonessential describe phenotypes, specifically whether a given gene is required for growth under a specific condition. Most viruses are finely tuned through selection to fit a specific niche. In some cases, not all viral genes are absolutely required for virus replication in that niche, particularly for viruses with large genomes. Furthermore, if the niche is changed—such as from a natural animal host to a cell line in a laboratory—some genes that may have been essential for productive infection in the animal may not be required for replication in cell culture. Genes that are required for growth under a specific condition are termed essential, and those that are not required are termed nonessential. Because as a phenotype essentiality may be a function of the specific test conditions, the test conditions need to be specified in describing the mutation. As an example, the herpesvirus thymidine kinase gene is nonessential for virus replication in cell culture. Genes that are either essential or nonessential under a given condition present unique characteristics for analysis. Thus, mutants in nonessential genes may be easy to isolate because the gene can be deleted, although the function of the gene may be difficult to determine because, by definition, nonessential genes have no phenotype. Conversely, genes that are essential can be used to study gene function by characterizing the precise replication defect caused by a mutation in the gene; however, acquiring the appropriate mutant is confounded by the necessity for identifying a condition that will permit growth of the virus for study. Generally, viruses with small genomes have few if any nonessential genes. For example, poliovirus encodes just 11 proteins, and all are essential for complete viral replication in cultured cells.

Loss of Function and Gain of Function Mutations that alter the function of a gene can be subclassified into two categories, loss of function or gain of function. Both loss-of-function and gain-of-function mutations are useful tools in virology. For loss-of-function mutations, the gene product lacks a molecular function of the wildtype gene. The inactivation of the viral gene product may be partial or complete. Examples of loss-of-function mutations include lack of vaccinia virus replication in human cells from K1L gene deletion,124 and lack of herpes simplex virus latency reactivation from viral thymidine kinase gene deletion.16 Conversely, for gain-of-function mutations, the gene product’s effect gets stronger or may even acquire different or abnormal functions. Examples of gain-of-function mutations include point mutations in influenza virus HA protein that increase transmission67,74 and a coxsackievirus capsid mutation that increases viral replication speed in cultured cells.96 Notably, there are often fitness “tradeoffs” for gain-offunction mutants, where fitness gain in one environment confers fitness loss in a different environment.35

Study of Mutations and Mutants Spontaneous Mutation Spontaneous mutation rates in viruses are measured by fluctuation analysis,77 a technique pioneered by Luria and Delbruck101 for analysis of mutation in bacteria, and later adapted to viruses by Luria.99 Fluctuation analysis consists of measuring the proportion of spontaneous mutants with a particular phenotype in many replicate cultures of virus and applying the Poisson distribution to these data to calculate a mutation rate.

Both DNA and RNA viruses undergo spontaneous mutation; however, the spontaneous mutation rate in RNA viruses is usually much higher than in DNA viruses. In general, the mutation rate at a specific site in different DNA viruses ranges from 10−8 to 10−11 per replication, whereas in RNA viruses, it is at least hundred-fold higher, between 10−3 and 10−6 per replication. The difference in mutation rate observed between RNA and DNA viruses is primarily from differences in the replication enzymes. Specifically, the DNA-dependent DNA polymerases used by DNA viruses contain a proofreading function, whereas the reverse transcriptases used by retroviruses and RNA-dependent RNA polymerases used by RNA viruses lack a proofreading function. The difference in spontaneous mutation rate has profound consequences for both the biology of the viruses and for laboratory genetic analysis of viruses. Specifically, RNA viruses exist in nature as quasispecies34—that is, populations of virus variants in relative equilibrium with the environment but capable of swift adaptation owing to a high spontaneous mutation rate. Conversely, DNA viruses are genetically more stable but less adaptable. In the laboratory, the high mutation rate in RNA viruses presents difficulties in routine genetic analysis because mutants easily revert to wild-type virus that can outgrow the mutant virus.

It is noteworthy that whereas the actual mutation rate at a single locus is probably relatively constant for a given virus, the apparent mutation rate to a given phenotype depends on the nature of the mutation(s), which can give rise to that phenotype. For example, spontaneous mutation to bromodeoxyuridine (BrdU) resistance in vaccinia virus may occur at least 10-100 times more frequently than spontaneous reversions of temperature-sensitive mutations to a wild-type, temperature-insensitive phenotype. In the case of BrdU resistance, any mutation that inactivates the thymidine kinase causes resistance to BrdU, and thus, there are literally hundreds of different ways in which spontaneous mutation can give rise to BrdU resistance. By contrast, a temperature-sensitive mutation is usually a single-base missense mutation, in which may exist only one possible mutational event that could cause reversion to the wild-type phenotype; thus, the apparent spontaneous mutation rate for the revertant phenotype is lower than the apparent spontaneous mutation rate to the BrdU-resistant phenotype. From a practical perspective, the apparent spontaneous mutation rate for specific selectable phenotypes may be sufficiently high such that induction of mutants is unnecessary for their isolation. However, for mutants where the desired mutational events are rare and a screen must be used rather than a selection (eg, temperature-sensitive mutants), induced mutation may be required for efficient isolation of mutants. Induced mutation is particularly useful for viruses with low error rates, such as many DNA viruses.

Induced Mutation While many RNA viruses have mutation frequencies high enough for relatively straightforward isolation of viral mutants, the incidence of spontaneous mutations in many DNA viruses is low enough that induction of mutation is a practical prerequisite. It is usually desirable to induce limited single-base changes, and for this purpose, chemical mutagens are most appropriate. Commonly used chemical mutagens are of two types: in vitro mutagens and in vivo mutagens.36 In vitro mutagens work by chemically altering nucleic acid and can be applied by treating virions in the absence of replication. Examples of in vitro mutagens include hydroxylamine, nitrous acid, and alkylating agents, which through chemical modification of specific bases cause mispairing leading to missense mutations. In vivo chemical mutagens comprise compounds such as nucleoside analogs that must be incorporated during viral replication and thus must be applied to an infected cell. One of the most effective mutagens is the alkylating agent nitrosoguanidine, which although is capable of alkylating nucleic acid in vitro is most effective when used in vivo, where it works by alkylating guanine residues at the replication fork, ultimately causing mispairing. The effectiveness of a mutagenesis is often assayed by observing the killing effect of the mutagen on the virus. Overall, the use of mutagens can increase the mutation frequency several hundred-fold, such that desired mutants may comprise as much as 0.5% of the total virus population.

RNA viruses have relatively high mutation frequencies, and increasing their mutation frequencies even more with mutagen treatment can 40

extinguish populations through “lethal mutagenesis.” For example, poliovirus propagated in the presence of the mutagenic nucleoside analog ribavirin has an increased mutation frequency, which can dramatically reduce viral fitness.25 Using mutagenic nucleoside analogs to induce lethal mutations in RNA viruses has been applied to several systems, ranging from HIV to coronaviruses.123,150

Double Mutants and Siblings The existence of double mutants and siblings can theoretically complicate genetic analysis of a virus. A double (or multiple) mutant is defined as a virus that contains more than one mutation contributing to a phenotype. Theoretically, because the probability that a double mutant will be created increases as the dose of a mutagen is increased, there is a practical limit to the amount of induced mutation that is desirable. Double mutants are usually revealed as mutants that are noncomplementing with more than one mutant or are impossible to map by recombination or physical methods. Siblings result from replication of mutant virus either through amplification of a mutagenized stock or during an in vivo mutagenesis. The only completely reliable method to avoid isolation of sibling mutants is to isolate each mutant from an independently plaquepurified stock of wild-type virus.

Mutant Genotypes There exist two basic categories of mutation: base substitution and deletion/insertion mutations. Both mutation types can occur with consequence in either a protein coding sequence or in a control sequence, such as a transcriptional promoter, a replication origin, or a packaging sequence. Base substitution mutations consist of the precise replacement of one nucleotide with a different nucleotide. In coding sequences, base substitution mutations can be silent, causing no change in amino acid sequence of a protein; they can be missense, causing replacement of the wild-type amino acid with a different residue; or they can be nonsense, causing premature translation termination during protein synthesis. Deletion and insertion mutations comprise deletion or insertion of one or more nucleotides in a nucleic acid sequence. In a coding sequence, deletion or insertion of multiples of three nucleotides can result in precise deletion or insertion of one or more amino acids in a protein sequence. In a coding sequence, deletions or insertions that do not involve multiples of three nucleotides result in a shift in the translational reading frame, which almost invariably results in premature termination at some distance downstream of the mutation. In general, nonsense mutations, frame shift mutations, or large in-frame insertions or deletions are expected to inactivate a gene, whereas missense mutations may cause inactivation or much more subtle phenotypes such as drug resistance or temperature sensitivity.

Mutant Phenotypes In the context of experimental virology where a goal is to understand the function of individual virus genes, mutants that inhibit virus replication by inactivating a viral gene are useful. The nonproductive infections with these lethal mutants can be studied in detail to determine the precise aspect of virus replication that has been affected, thus providing information about the normal function of the affected gene. However, one must be able to grow the mutant to conduct experiments. Thus, a condition must be found where the mutation in question is not lethal—hence, the general class of mutant phenotypes, conditional lethal. Conditional lethal mutants are one of the most useful classes of mutant phenotypes, consisting of host-range, nonsense, temperature-sensitive, and drug-dependent phenotypes, described individually in the next section. Two additional classes of mutant phenotypes—resistance and plaque morphology—have very specific application to genetic analysis of viruses and are also described. Host range A host-range virus mutant is broadly defined as a mutant that grows on one cell type and not on another, in contrast to wild-type virus, which grows on both cell types. Two general subcategories of host-range mutants exist: natural and engineered. Natural host-range virus mutants are relatively rare, primarily because they must be identified by brute force screen or serendipity. The existence of a host-range phenotype implies that a specific virus-host interaction is compromised, which also implies that for any specific host-range phenotype, only one or a limited number of virus genes will be targeted. A classic example of a natural host-range mutant is the host range-transformation (hr-t) mutants of mouse polyoma virus, which affect both small and middle T antigens and grow on primary mouse cells but not continuous mouse 3T3 cell lines.5 Engineered host-range mutants are constructed by deleting an essential gene of interest in the virus while at the same time creating a cell line that expresses the gene. The engineered cell line provides a permissive host for growth of the mutant virus because it complements the missing virus function, whereas the normal host lacking the gene of interest provides a nonpermissive host for study of the phenotype of the virus. This technology has been useful for study of a variety of viruses, notably adenovirus and herpes simplex virus, where it has facilitated study of several essential virus genes.29,79

Nonsense mutants Nonsense mutants contain a premature translation termination mutation in the coding region of the mutant gene. They are formally a specific class of conditionally lethal, host-range mutants. Specifically, the permissive host is one that expresses a transfer RNA (tRNA) containing an anticodon mutation that results in insertion of an amino acid in response to a nonsense codon, thus restoring synthesis of a full-length polypeptide and suppressing the effects of the virus nonsense mutation. The nonpermissive host is a normal cell in which a truncated, nonfunctional polypeptide is made. In practice, most nonsense mutants in existence have been isolated by random mutagenesis followed by a brute force screen for host range. Nonsense mutants have three distinct advantages for the conduct of virus genetics: (1) mutants can be isolated in virtually any essential virus gene using one set of permissive and nonpermissive hosts and one set of techniques; (2) the mutations result in synthesis of a truncated polypeptide, thereby facilitating identification of the affected gene; and (3) virus mutants can be engineered relatively easily because the exact sequence of the desired mutation is predictable. Nonsense mutants have provided the single most powerful genetic tool in the study of bacteriophage, where efficient, viable nonsense suppressing bacteria are readily available. Unfortunately, attempts to isolate nonsense-suppressing mammalian cells have met with only limited success, probably because the nonsense-suppressing tRNAs are lethal in the eukaryotic host.118,144

Temperature sensitivity Temperature sensitivity is a type of conditional lethality in which mutants can grow at a low temperature but not a high temperature, in contrast to wild-type virus, which grows at both temperatures. Genotypically, temperature-sensitive mutations result usually from relatively subtle single 41

amino acid substitutions that render the target protein unstable and hence nonfunctional at an elevated or nonpermissive temperature while leaving the protein stable and functional at a low, permissive temperature. Additionally, mutations in noncoding regions can confer temperature sensitivity, likely through effects on RNA structure.140 Temperature-sensitive mutants are usually isolated by random mutagenesis followed by brute force screening for growth at two temperatures. Screening can be streamlined by a plaque enlargement technique in which mutagenized virus is first plated at a permissive temperature, then stained and shifted to a nonpermissive temperature after marking the size of plaques, to screen for plaques that do not increase in size at the nonpermissive temperature.149 Replica plating techniques that permit relatively straightforward screening of thousands of mutant candidates in yeast and bacteria have not been successfully adapted to virology; thus, a screen for temperature sensitivity, even when streamlined with plaque enlargement, ultimately depends on the laborious but reliable process of picking and testing individual plaques. Temperature-sensitive mutants have the profound advantage of theoretically accessing any essential virus gene using a single set of protocols. Temperature-sensitive mutants have proved enormously useful in all branches of virology but have been particularly useful for the study of animal viruses, where nonsense suppression has not been a viable option. Cold-sensitive mutants (ie, mutants that grow at a high but not a low temperature) comprise a relatively rare but nevertheless useful alternate type of temperature-sensitive mutants.

Drug resistance and dependence Several antiviral compounds have been identified, and virus mutants that are resistant to or depend on these compounds have been useful in genetic analysis of viruses. A few compounds have been identified that target similar enzymes in different viruses, including phosphonoacetic acid, which inhibits DNA polymerases,64,152 and BrdU, which targets thymidine kinases.38,156 More often, however, antiviral drugs are highly specific for a gene product of one particular virus—for example, guanidine, which targets the polio 2C NTPase128; acyclovir, which targets the herpes simplex virus thymidine kinase and DNA polymerase17,143; amantadine, which targets the influenza virus M2 virion integral membrane ion channel protein63; or isatin-β-thiosemicarbazone, which is highly specific for poxviruses and targets at least two genes involved in viral transcription.19,24 The most useful drugs are those that inhibit wild-type virus growth in a plaque assay without killing cells in a monolayer, such that resistant or dependent viruses can be selected by virtue of their ability to form plaques on a drug-treated monolayer.

Drug-resistant or drug-dependent virus mutants have two general uses in virus genetics. First, they can be useful in identifying the target or mechanism of action of an antiviral drug. For example, studies of influenza virus mutants resistant to amantadine were of importance in characterizing both the M2 gene and the mechanism of action of amantadine.129 Similarly, viruses resistant to the mutagenic nucleoside analog ribavirin have polymerase mutations that increase fidelity of RNA virus replication, further supporting viral mutagenesis as an antiviral mechanism.25,126 Second, resistant or dependent mutants provide selectable markers for use in recombination mapping, for the assessment of specific genetic protocols, or for selection of recombinant viruses in reverse genetic protocols. For example, guanidine resistance has been used as a marker for use in three-factor crosses in recombination mapping of poliovirus temperature-sensitive mutants23; phosphonoacetic acid resistance and isatin-β-thiosemicarbazone dependence have been used in vaccinia virus to assess the efficiency of marker rescue protocols,43 and acyclovir resistance and BrdU resistance, resulting from mutation of the herpesvirus or poxvirus thymidine kinase genes, have been used in both herpesviruses and in poxviruses to select for insertion of engineered genes into the viral genome.18,105,121

Plaque morphology Plaque morphology mutants are those in which the appearance of mutant plaques is readily distinguishable from wild-type plaques. Most commonly, the morphological distinction is plaque size (ie, mutant plaques may be larger or smaller than wild-type plaques); however, other morphological distinctions are possible, such as formation of clear vs turbid bacteriophage plaques. Most plaque morphology mutants affect very specific virus functions, which in turn affect the virus-host relationship in a fashion that impacts on the appearance of a plaque. Notable examples from bacteriophage research include clear plaque mutants of bacteriophage lambda and rapid lysis mutants of the T-even bacteriophage. Wild-type lambda forms turbid plaques because some percentage of cells are lysogenized and thus survive the infection, leaving intact bacteria within a plaque. Clear mutants of lambda typically affect the lambda repressor such that lysogeny is prevented and all infected bacteria lyse, resulting in a clear plaque.81 Wild-type T-even phages produce small plaques with a turbid halo because only a fraction of infected bacteria lyse during a normal infection, a phenomenon called lysis inhibition. Rapid lysis mutants, which affect a phage membrane protein, do not display lysis inhibition and as a result form large, clear plaques.68 Examples from animal virus research include large plaque mutants of adenovirus and coxsackievirus, and syncytial mutants of herpes simplex virus. The large plaque phenotype in adenovirus results from faster than normal release of virus from infected cells,86 and the large plaque phenotype in coxsackievirus results from reduced binding to inhibitory glycans.170 Syncytial mutants of herpesvirus express altered virus surface glycoproteins and result in fusion of infected cells, whereas wild-type virus infection causes cells to round and clump without significant fusion. Thus, syncytial mutants form large plaques readily distinguishable from the smaller dense foci caused by wild-type virus.137 All of these specific plaque morphology mutants have value either in the study of the actual functions affected or as specific phenotypic markers for use in recombination studies, where they can be used in the same fashion as drug resistance markers.

In addition to the existence of specific plaque morphology loci in several viruses, it is noteworthy that any mutation that affects virus yield, growth rate, or spread may result in production of a smaller than wild-type plaque, which can be useful in genetic experiments. Thus, many temperature-sensitive mutants form smaller than wild-type plaques even at the permissive temperature because the mutant gene may not be fully functional even under permissive conditions, and this property is often useful in mutant isolation or for distinguishing wild-type from mutant virus in plaque assays involving several virus variants. Finally, unique functions of specific viral proteins can alter plaque morphology. Vaccinia virus encodes two proteins, A33 and A36, that are expressed on the cell surface early after infection and “mark” the cell as infected.31 As a consequence, superinfecting virions are repelled and available to infect other uninfected cells, enabling spread. Mutant viruses lacking expression of these proteins have small plaque phenotypes.

Neutralization escape Neutralization escape mutants are a specific class of mutants selected as variant viruses that form plaques in the presence of neutralizing antibodies. Such mutants affect the structure or modification of viral surface proteins and have been of value in studies of virus structure, antigenic variation, and virus-cell interactions.55,70 Neutralization escape has been particularly well studied during the SARS-CoV-2 pandemic given its practical relevance for immune evasion and spread of viral variants.172 42

Reversion Reversion may be defined as mutation that results in a change from a mutant genotype to the original wild-type genotype. Accordingly, revertants in a stock of mutant virus are revealed as viruses that have acquired a wild-type phenotype. Spontaneous reversion of missense mutations probably results from misincorporation during replication, because the reversion frequency of different viruses often reflects the error rate of the replication enzyme. Spontaneous reversion of significant deletion mutations occurs rarely, if at all, because reversion would require replacement of missing nucleotides with the correct sequence. Reversion impacts on viral genetics in two ways. First, in any genetic experiment involving mixed infections with two genetically different viruses, wild-type viruses can arise either through reversion or recombination; in most cases, it is important to be able to distinguish between these two processes. This is discussed in more detail in the later sections describing complementation and recombination. Second, as described earlier in the description of spontaneous mutation, if the spontaneous reversion rate is extremely high, revertants can easily come to dominate a mutant virus stock, thus obscuring the mutant phenotype and causing serious difficulties in both genetic and biochemical analysis of mutants.

Leakiness Not all conditionally lethal mutants are completely defective in replication under nonpermissive conditions, and leakiness is a quantitative measure of the ability of a mutant virus to grow under nonpermissive conditions. Leakiness can be quantified with a one-step growth experiment. To quantify leakiness of a temperature-sensitive mutant, for example, cells are infected at a high MOI with wild-type or mutant virus, infected cells are incubated at either permissive or nonpermissive temperatures, and maximum virus yields are then determined by plaque titration under permissive conditions so that the growth of mutant and wild-type virus can be quantitatively compared. Ideally, for wild-type virus, the ratio of the yield for infections done at the nonpermissive temperature relative to the permissive temperature should be one—that is, the virus should grow equally well at both temperatures. For mutant viruses, the ratio of the yield for infections done at the nonpermissive temperature relative to the permissive temperature may range from 100-fold in some cell types.240 Nevertheless, its toxicity in particular cells is a serious clinical issue. In particular, AZT causes bone marrow suppression that manifests most commonly as neutropenia and anemia.370 AZT toxicity appears to be due not only to the effects of AZT-TP on cellular polymerases but also to the effects of AZT-monophosphate, which is both a substrate for cellular thymidylate kinase and an inhibitor of this essential enzyme.140 For a number of other anti-HIV nucleoside analogs, a key determinant of toxicity appears to be the mitochondrial DNA polymerase (DNA polymerase γ61,249,285).

AZT-resistance mutations accumulate in the HIV pol gene that encodes RT43,77,212,242 (Fig. 14.9). Interestingly, four or more mutations are required to confer high-level resistance, but some of the mutations confer little or no resistance on their own.212,241,242 These latter mutations appear to be selected because they boost resistance by the other mutations or compensate for fitness costs of certain mutations. The requirement for multiple mutations for high-level resistance likely explains the relatively slow emergence of AZT resistance in patients and during selection in cell culture.145,238 Based on sequencing of virus isolated from patients following cessation of AZT therapy or in individuals newly infected with resistant virus, and of virus from replication competition experiments in vitro, AZT-resistant mutants appear to be modestly less fit than wild-type virus.97

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FIGURE 14.9 Locations of drug resistance alterations in the HIV-1 and HBV polymerases. The HIV-1 reverse transcriptase (RT) (top) and the HBV DNA polymerase (bottom) are related enzymes with sequence and predicted structural homology and are cartooned as bars. The HBV enzyme has a segment that is unrelated to the HIV enzyme, which is depicted here as looping out. The positions of selected substitutions and insertions that confer drug resistance are indicated in single letter code with the wild-type residue above the bar, the residue number within the bar, and mutant residues or inserts below the bar. Methionine 184 in HIV RT and methionine 204 in HBV polymerase are embedded within a YMDD motif (green) that is important for polymerase catalysis. Alterations in this methionine confer resistance to 3TC and related L-stereoisomer drugs, as does alteration of alanine 181 in HBV polymerase (also green). Boldfaced residues in HIV RT indicate the positions of mutations that affect susceptibility to most anti-HIV nucleoside analogs. An insertion at residue 69 in HIV RT (blue) together with other alterations including M41L, K70R, L210W, T215Y, and K219Q also affect susceptibility to these drugs. Substitution of arginine for lysine 65 (purple) in HIV RT confers resistance to all nucleoside analogs except AZT. Substitutions of lysine 103 and tyrosine 181 (italics) confer resistance to nonnucleoside reverse transcriptase inhibitors. Alterations of isoleucine 169, threonine 184, serine 202, or methionine 250 (orange) together with a leucine to methionine substitution at residue 180 (orange) plus a methionine 204 substitution in HBV polymerase can result in entecavir resistance. A threonine for asparagine 236 substitution in HBV polymerase (blue) can result in resistance to adefovir (high-level) and tenofovir (intermediate). The mechanism of AZT resistance stems from chain elongation by 3′-5′ phosphodiester bond formation being a reversible process, so that in the presence of pyrophosphate or ATP, RT can catalytically excise the terminal nucleotide by a process termed pyrophosphorolysis (Fig. 14.10).12,302 Given the Km's of pyrophosphate and ATP for this reaction, and the concentrations of these species in cells, ATP is the likely substrate in vivo. The viral DNA chain is now free to resume elongation with the removal of the chain terminating nucleoside analog. AZT resistance mutations facilitate this reverse reaction of excision repair.252,303 Crystal structures of wild-type and AZT-resistant RTs bound to either AZT-TP or the excision product indicate that resistance mutations alter the way that the AMP moiety of the excision product binds without affecting the AZT-TP–binding site.443

FIGURE 14.10 Biochemical mechanism of resistance to AZT. During synthesis of HIV DNA by HIV RT, AZT-TP is incorporated into the growing primer strand (primer-p-AZT), generating pyrophosphate (PPi). This reaction is reversible, so that RT can excise AZTMP from the primer terminus and combine it with PPi, regenerating AZT-TP and primer/template. ATP can substitute for PPi in this excision reaction by donating two phosphates to AZT-MP. The excision reaction is enhanced by AZT resistance mutations but impaired by NNRTIs and certain HIV RT substitutions that confer resistance to 3TC (eg, M184V) and NNRTIs (eg, Y181C). This resistance mechanism of excision repair applies to most, if not all anti-HIV nucleoside analogs; thus, these mutations have been termed nucleoside-associated mutations (NAMs) and account for many examples of cross resistance of HIV toward various nucleoside analogs.462 Regardless of mechanism, various combinations of mutations affecting HIV RT can confer resistance to every approved nucleoside analog (Fig. 14.9; Ref.207).

The limited clinical effectiveness of AZT and problems with toxicity and resistance led to the development of other anti-HIV drugs and the use of combination chemotherapy.

Lamivudine and Emtricitabine 434

Lamivudine (3TC) was reported to exhibit anti-HIV activity in 1992.78,79,391 Lamivudine and its close relative emtricitabine (FTC) appear to exhibit the least toxicity of the anti-HIV nucleoside analogs. This may be related to their unusual sugar moiety that contains a sulfur atom and is an l-stereoisomer, not the standard d-stereoisomer of normal nucleosides (Fig. 14.7C). Lamivudine is sequentially phosphorylated by cellular enzymes to its triphosphate.54 Like AZT-triphosphate, 3TC-triphosphate is both a competitive inhibitor and a substrate for RT, and once incorporated, is an obligate chain-terminator of DNA synthesis. It is a more potent inhibitor of HIV RT than cellular polymerases.171 The relative lack of toxicity of 3TC may be due to the negligible inhibition of host polymerases including mitochondrial DNA polymerase by 3TCtriphosphate.171

High-level resistance to 3TC develops rapidly both in cell culture and in patients treated with this drug alone (reviewed in Ref.100). A single mutation at codon 184 from Met to Val (M184V) or to Ile (M184I) confers a high degree of resistance to 3TC. M184 is within the conserved YMDD motif, in which the two Asp (D) residues are involved in catalysis of polymerization (Fig. 14.9). Both mutants are less fit than wild-type virus, which may be due to decreased processivity, primer usage, or initiation by the mutant enzyme or some combination of these (reviewed in Ref.100). The crystal structure of a 3TC-resistant RT was used to develop a molecular model that posits that codon 184 mutations result in steric hindrance that obstruct incorporation of 3TC-triphosphate, but not normal nucleotides.386

The M184V mutation has a number of other interesting effects, including conferring low-level resistance to some (eg, abacavir, Fig. 14.6) or hypersensitivity to other nucleoside analogs (eg, AZT and tenofovir; reviewed in Ref.100). This hypersensitivity to AZT and other drugs in the absence of other mutations is also observed in the presence of known drug resistance mutations such that levels of resistance are reduced with the addition of M184V. This phenotypic suppression is best explained by the M184V mutation reducing the excision repair of AZT-terminated primers (Fig. 14.10; Ref.100). Nevertheless, an accumulation of multiple mutations can result in high-level resistance to both AZT and 3TC, and other single mutations such as K65R can confer resistance to 3TC (and most other nucleoside analogs) (Fig. 14.9; Ref.304).

The mechanisms of action and resistance of FTC are very similar to those of 3TC; however, it may be slightly more potent and have a longer intracellular half-life.369 Its coformulation with tenofovir (see below) has made this combination a backbone of combination antiretroviral therapy.

Other Anti-HIV Nucleoside Analogs There have been numerous other anti-HIV nucleoside analogs approved over the years, but several of them have been discontinued. Two that are still used are abacavir and tenofovir. Abacavir contains an altered guanosine and an altered sugar that lacks both 2′ and 3′ OH and the ether oxygen (Fig. 14.7C). Following uptake into cells, abacavir is phosphorylated to its monophosphate, and then the 6-cyclopropylamino alteration on the base is removed, resulting in guanine linked to the modified sugar (carbovir), followed by further phosphorylation to the triphosphate.124 Abacavir is also notable as an example of the use of pharmacogenomic tests to exclude patients likely to suffer adverse events, in this case those with the HLA-B*-5701 allele, which predisposes to a severe hypersensitivity reaction.276 Tenofovir is an acyclic deoxyadenosine monophosphate analog (ie, a nucleotide analog) lacking a 3′OH and the entire 2′ moiety (Fig. 14.7C). It is administered as an orally available prodrug, either tenofovir disoproxil or tenofovir alafenamide (structures of these prodrugs not shown). These formulations have the advantage of being administered just once daily, making them attractive as constituents of long-term combination therapies.

Anti-HBV Nucleoside Analogs Five nucleoside analogs—lamivudine (3TC), tenofovir, telbivudine, adefovir, and entecavir—have been approved for the treatment of HBV. Lamivudine and tenofovir have been described above under anti-HIV nucleoside analogs. Telbivudine is simply l-thymidine, thus sharing this isomeric configuration with 3TC and FTC, but has been withdrawn so will not be discussed further. Adefovir, like tenofovir, is a nucleotide analog with an adenine linked to an altered sugar similar to that in acyclovir, but containing an additional methylene moiety (Fig. 14.7C). Also, like tenofovir, it is administered as an orally available prodrug, in this case as adefovir dipivoxil. Entecavir is an unusual deoxyguanosine nucleoside analog in which the ether oxygen of the sugar is replaced with an exo carbon-carbon double bond (Fig. 14.7C). The mechanisms of action of anti-HBV nucleoside analogs are very similar to those of anti-HIV nucleoside analogs: They are converted to triphosphates by cellular enzymes, are incorporated into the growing DNA chain by HBV DNA polymerase (also a reverse transcriptase), and cause chain termination. Notably entecavir is a nonobligate chain terminator. Termination occurs two or three bases after its incorporation,392 but precisely how this occurs remains unclear. Of note, entecavir-triphosphate has a higher affinity for HBV polymerase than does dGTP.392

The similarities in mechanisms of action of these drugs against the two viruses extend to some similarities in mechanisms of resistance but also important differences.270 For example, a mutation in the methionine codon in the HBV pol gene that corresponds to codon M184 in the HIV pol gene can confer high-level resistance to 3TC (Fig. 14.9); however, in this case, the methionine to isoleucine mutant retains fitness, whereas methionine to valine or serine substitutions are usually only found in the presence of additional mutations. There is little if any evidence for HBV drug resistance mutations resulting in increased excision of incorporated drug as do AZT resistance mutations of HIV; rather, the mutations appear to affect incorporation of drug-triphosphate by various mechanisms. Interestingly, drug resistance that requires only a single mutation develops relatively quickly, but still more slowly in vivo with HBV than with HIV. In at least some cases, this may be because sites of mutation also encode residues in an overlapping reading frame that affect HBV surface antigen.

As is the case with AZT resistance, multiple mutations are required to confer high-level resistance to entecavir, and some of these mutations confer little resistance on their own.270 This correlates with slow development of resistance in vivo. The greater potency and slower rates of resistance to entecavir and tenofovir has led to these drugs becoming preferred for treatment of HBV.270 FTC and tenofovir are often used in patients who are infected with both HIV and HBV, because they are active against both viruses (even though FTC is not FDA approved for treating HBV infections).

Anti-RNA Virus Nucleoside Analogs With all the nucleoside analog drugs against DNA viruses and retroviruses, it may seem surprising that there are so few anti-RNA virus nucleoside analogs. One possible contributor to this outcome may be the higher concentrations of competing ribonucleotides in cells relative to deoxyribonucleotides. Nevertheless, there is one long-standing nucleoside analog, ribavirin, that was approved for use against certain RNA 435

viruses; sofosbuvir, which is a mainstay anti-HCV drug; and now, two new ones, molnupiravir (see above) and remdesivir, which have received emergency authorization against SARS-CoV-2.

Ribavirin Ribavirin is a nucleoside analog unlike the other nucleoside analogs discussed thus far in that it has a normal D-stereoisomer sugar (ribose) attached to a baselike moiety (Fig. 14.7D). It exhibits activity against many viruses in cell culture, but it has been approved only as monotherapy for severe respiratory syncytial virus (RSV) infections, and in combination with interferons for chronic HCV infections. Its routine use against RSV is not recommended except in certain complicated cases,444 and its use against HCV has been superseded by newer, much more effective drugs. In both cases, it is not clear if it exhibits meaningful antiviral activity in patients.41,99 Moreover, even in cell culture, its antiviral mechanism of action against RSV and HCV is still not well understood with multiple mechanisms proposed, including “error catastrophe,”106 which, as discussed above, is a leading hypothesis for the mechanism of molnupiravir. However, in HCV replicons, ribavirin resistance was found to be due to mutations affecting a protein other than polymerase,348 and studies of viral mutagenesis in patients treated with ribavirin have not provided definitive answers.277,345 Further investigations of the mechanisms of ribavirin action and resistance may yield interesting new insights into virus biology and biochemistry.

Sofosbuvir, an Anti-HCV Nucleoside Analog Sofosbuvir is a prodrug of β-D-2′-deoxy-2′-R-fluoro-2′-β-C-methyluridine monophosphate. Its corresponding nucleoside was initially identified as a metabolite of another drug313,407 but did not show appreciable activity in cells, potentially due to slow formation of the nucleoside monophosphate.275,407 Compound optimization efforts, therefore, focused on cell permeable prodrugs of the nucleoside monophosphate that could be selectively activated in the liver to mitigate potential off target toxicity.141,407 These efforts lead to the discovery of the phenoxy phosphoramidate prodrug sofosbuvir407 (Fig. 14.7D).

Postcellular uptake, the prodrug is cleaved by either carboxyesterase 1, or cathepsin A, and subsequently undergoes an intermolecular attack to release phenol.314 Generation of the active nucleoside triphosphate is completed through hydrolysis of the alaninyl moiety to generate the monophosphate by histidine triad nucleotide-binding protein1 (Hint1), followed by di- and triphosphorylation by uridine/cytidine monophosphate kinase nucleotide diphosphate kinase, respectively.141,314 Biochemical studies demonstrated sofosbuvir triphosphate is a selective substrate/inhibitor of the HCV RNA–dependent RNA polymerase vs human DNA and RNA polymerases.11,235,275,313 Following incorporation into the growing RNA strand, sofosbuvir acts as a nonobligate chain terminator, terminating extension without addition of other nucleotides,275 but how it induces termination is unclear. In cellular systems, sofosbuvir demonstrates similar activity across genotypes with 20-40 nM EC50 values in genotype 1-3 replicon assays.234,235,374 In vitro resistance selection with sofosbuvir and nucleosides with a similar structure identified S282T mutation within the polymerase, which causes a 10-fold decrease in replicon sensitivity to sofosbuvir in the genotype 1 background.234 A more complex resistance picture emerged upon resistance selection in the genotype 2 background where T179A, M289L, I293L, M434T, and H479P mutation were observed in addition to S282T; these mutations in combination with S282T were required to generate significant resistance to sofosbuvir.234 Decreased replication fitness was observed with the S282T mutation across genotypes.234 Treatment emergent clinical resistance to sofosbuvir is extremely rare consistent with a high barrier to resistance.244,426 Sofosbuvir is a component of several HCV therapies (see under Therapy of Viral Hepatitis).

Remdesivir, an Anti–SARS-CoV-2 Nucleoside Analog Remdesivir is a prodrug of 1′cyano 4-aza-7,9-dideazaadenosine (Fig. 14.7D). The compound was initially identified by examining a series of modified 1′-substituted 4-aza-7,9-dideazaadenosine C-nucleosides.69 To improve cell permeability and potency, the nucleoside was converted to the monophosphoramidate prodrug.69,457 Subsequently, remdesivir demonstrated 8 years old), they raise the possibility that without mitigation, further agriculturally mediated outbreaks of BSE or transmissions to other species might occur. The feed-mediated amplification of C-BSE implies that most cases were due to oral exposures. Oral dosing experiments indicated that transmission of C-BSE to cattle can require as little as 1 mg of infected brain tissue.207 After oral exposure, infectivity can be detected early in the ileal Peyer patches and then progress primarily via the autonomic nervous system to the brain prior to the onset of symptomatic disease.3,177 In later stages, the infection disseminates further to other tissues including muscles.3,26,107 Accordingly, some countries have designated multiple tissues as specified risk materials (SRMs) that should be excluded from human food, pharmaceuticals, and animal feeds to reduce the risk of transmission.

The atypical forms of BSE were discovered during surveillance of healthy and fallen stock. The designations of H- and L-BSE (or BASE) are based on the relative sizes (higher vs lower) of protease-treated PrPRes bands on immunoblots.105,107,177 Although the causes of these atypical types of BSE remain uncertain, the rarity and age-dependence of their incidence are consistent with spontaneous, rather than acquired, etiologies. Both L- and H-BSE are transmissible as distinct prion strains via intracerebral inoculation,30,62,208,236 but transmission of H-BSE to mice expressing bovine PrP has sometimes produced a C-BSE-like strain.384 Oral transmission of L-BSE to cattle has been inefficient, with a low clinical attack rate.290 L- and H-BSE PrPd are found most abundantly in the central and peripheral nervous systems with little accumulation in lymphoid organs (reviewed in Refs.107,177,215).

Scrapie in Small Ruminants As with BSE, scrapie strains in sheep have been described as “classical” or “atypical” with the latter referring to the Nor98 strain.38,70,177,257 The occurrence of scrapie in small ruminants extends to many parts of the world, including 25 countries in the EU, with a prevalence of 9 and 6 cases per 10 000 tested animals for typical and atypical scrapie, respectively.177 Interestingly, atypical scrapie also has been detected in Australia and New Zealand, countries that have been regarded as free of classical scrapie. The prevalence of classical scrapie varies within countries and over time, consistent with contagion between infected animals being the main etiology. In contrast, the prevalence of atypical scrapie is more consistent, regardless of exposure. Such findings support the notion that although atypical scrapie is experimentally transmissible, it is not as naturally contagious as classical scrapie and may be largely spontaneous.

Differences in the tissue distributions of classical and atypical scrapie PrPSc may help to explain the apparent differences in the natural contagiousness of these strains.70,177 In classical scrapie, the infection is usually contracted orally within the first weeks of life. The infection enters gut-associated lymphoid tissue such as the Peyer patches and then spreads to other lymphoid organs, and on to the spinal cord and brain via the autonomic nervous system. Eventually, the infection spreads centrifugally to other tissues such as the peripheral nervous system, muscle, and blood. Pregnant ewes can accumulate infectivity in their placentas, which is shed into the environment as the afterbirth. Infectivity is also found in colostrum and milk. As a result, perinatal exposures contribute significantly to the natural spread of classical scrapie within flocks.

The distribution of atypical scrapie prions is more restricted to the central nervous system, presumably limiting the potential for environment shedding or transmission via casual contact,177 but atypical scrapie can be transmitted experimentally via the oral route, raising the possibility that this might occur naturally to some extent.

Sheep have polymorphisms at PRNP codons 136, 154, and 171 that can influence susceptibility to scrapie (Fig. 22.13), with those homozygous for the A136, R154, R171 (ARR) allele being particularly resistant to scrapie.70,177 Indeed, the resistance of this sheep genotype to classical scrapie has been used as justification for breeding programs aimed at generating scrapie-resistant flocks. Such programs have markedly reduced the prevalence of scrapie in Europe15 and the United States.12 However, a note of caution is that carriers of the ARR allele are susceptible to atypical Nor98 scrapie.38 Also of concern is the emergence of a C-BSE–like prion strain in asymptomatic pigs inoculated with atypical (Nor98) sheep scrapie.258 Such findings emphasize that resistance of host genotypes to prions can be strain dependent and that transmissions into intermediate species can alter prion strain characteristics and, perhaps, zoonotic potential.

Chronic Wasting Disease Chronic wasting disease (CWD) is a prion disease of cervids that was first identified in 1967 in a captive mule deer in Colorado.415 Since that time, it has steadily spread, both through transport of infected captive deer and elk and through wild animal migration, to at least 26 U.S. states, 3 Canadian provinces, and has affected cervids in Finland, Norway, Sweden, and South Korea (https://www.cdc.gov/prions /cwd/occurrence.html). The cervids affected include deer, elk, moose, and reindeer. CWD is the most efficiently transmitted prion disease and is now the most prevalent prion disease, with cases continuing to rise. Up to 30% of animals are infected in some free-ranging herds, and rates approach 100% in some captive deer herds. Management has proven difficult because CWD is being spread throughout the extensive geographic ranges of wild deer and elk, and CWD prions cannot be easily cleared from the environment, where they avidly bind soil and likely remain infectious for years.216

As with typical scrapie, CWD transmission is largely by the oral route. Prions can be found in many tissues of CWD-affected cervids, including the central nervous system, lymphoid tissues, spleen and pancreas, peripheral nerves, muscle, blood, and antler velvet.129 Prions are shed into the environment through saliva, feces, urine, placenta, and carcasses, leading to environmental contamination and horizontal transmission.260 There is also evidence for vertical transmission.282,347 The fact that CWD prions are present in muscle10 and elk velvet,11 raise particular concern for human exposure and potential zoonotic transmission.

Several polymorphisms in the cervid PRNP gene have been linked to reduced CWD susceptibility, delayed disease onset, altered disease course, and/or pathology (Fig. 22.13).13,14,278,285 These include alleles encoding leucine (L) at residue 132 in North American elk, asparagine (N) at residue 138 in reindeer or caribou, phenylalanine (F) at residue 225 in mule deer, and serine (S) at residue 96 in whitetail deer. Early results from a breeding strategy to reduce the frequency of the highly susceptible 96G (glycine at residue 96) PRNP variant in farmed whitetail deer have been promising with respect to CWD prevalence.165

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Camel Prion Disease Recently, prion disease has been discovered in three dromedary camels (>8 years of age) brought to a slaughterhouse in Algeria.23 These animals had pathognomonic neurodegeneration and PrPd in brain tissue. The origin of camel prion disease is unknown, but detection of PrPd in lymphoid tissue increases the chances of horizontal spreading among individuals. A retrospective analysis indicated that 3.1% of camels coming to the slaughterhouse in 2015-2016 had neurological signs suggesting prion disease.

Other Mammalian Prion Diseases Prion diseases of humans, cattle, sheep, and cervids have been transmitted experimentally or inadvertently to a wide range of additional mammalian species including nonhuman primates, mink (transmissible mink encephalopathy), cats (feline spongiform encephalopathy), pigs, rodents, and exotic ungulates.4,353 Of note, some species are susceptible to an unusually broad range of prion strains, that is, bank voles287,403 and squirrel monkeys,56,259,321 while others are largely resistant, for example, dogs.281,396 However, it is not clear whether these additional species naturally harbor their own prion disorders. Gianluigi Zanusso and colleagues revealed an intriguing situation in which a man and his cat had simultaneous prion diseases, with the man’s disease like sCJD and the cat’s being distinct from the feline spongiform encephalopathy resulting from BSE infections.423 However, the origin(s) of these two cases remains unknown.

Given the diversity of prion strains in mammals, practical concerns include the extent to which interspecies exposures to prion-infected materials might generate prion strains with new transmission and virulence characteristics. Examples of such shifts in prion strain characteristics upon interspecies transmission are well documented.34,196,258 A related concern is whether CWD is transmissible from cervids into predators, scavengers, other wildlife, or domesticated species. This appears not to be the case for mountain lions at least.418 However, mountain lions, crows, and coyotes can shed prions that they have consumed in their feces for a few days, potentially contributing to environmental dissemination.37 On a positive note, the prion seeding activity shed from mountain lions is only ~3% of that consumed, indicating an ability to either degrade or sequester ingested CWD prions.

Detection of Prions The detection of prions, or surrogates thereof, is important in many facets of prion disease management, including surveillance, risk reduction, diagnosis, therapeutics, and fundamental research. Historically, prions have been detected by animal bioassays taking months to years. Much more rapid immunoassays, such as ELISA, Western blot, or immunohistochemistry, can now be used for prion (or PrPd) detection in specimens with high prion loads, such as nervous or lymphoid tissues.265 However, up to 10 million-fold higher sensitivities, even exceeding those of animal bioassays, can be obtained with cell-free prion seed amplification assays such as PMCA,334 amyloid seeding assay,99 or RTQuIC.22,135,265,410 Certain murine prions can also be assayed with high sensitivity using scrapie cell assays201 that involve prion amplification through multiple in vitro passages followed by in situ immune detection of infected cells.

Many adaptations of cell-free amplification assays have been developed for most mammalian prion strains.135,294 Although amplification assays are more time-consuming and technically demanding than ELISA assays, they provide the sensitivity necessary for detecting low levels of prion seeds in diagnostic specimens that can be collected from live individuals or released into the environment. As noted above, the most commonly used amplification assay platform for human diagnostics is RT-QuIC. Antemortem diagnostics for animal prion diseases are under development70,134,166,167 and, if sufficiently practical and validated, may become particularly important in the determination of prion infection status in cervids and sheep as part of surveillance and prion-free certification programs. One approach to testing for CWD in live cervids has been to analyze rectoanal mucosal lymphoid–associated tissue (RAMALT) biopsied from anesthetized animals.167 Several studies have demonstrated markedly improved diagnostic sensitivity when RAMALT biopsies are tested with RT-QuIC vs immunohistochemistry.166 Another promising but as yet unvalidated approach is RT-QuIC testing of outer ear punches.134

Antemortem diagnosis has not only been difficult historically for PrP-based prion diseases but also for many of the other human proteinopathies. Accordingly, RT-QuIC–like seed amplification assays are being developed for many of these other diseases as well.135

Inactivation of Prions Prions are often more resistant to inactivation than typical microbes and may resist decontamination procedures that are routine in clinical, agricultural, or mortuary settings. However, certain chemical treatments, prolonged high temperature autoclaving, and thorough incineration, alone or in combination, can be effective in decontaminating contaminated materials and surfaces. The reader is referred to the Biosafety in Microbiological and Biomedical Laboratories 6th edition for details and recommended handling protocols.76

Perspectives Major challenges remain to be tackled in the prion diseases field. We currently have three near-atomic structures of two rodent prion strains, but there are dozens more to be determined to fully understand prion diversity. Research into the pathogenesis of prions has been rich and multifaceted. However, it remains unclear how to prioritize the many ways that prions can be neurotoxic, and how to translate such information into practical therapeutics. Although prion disease diagnostics in living human patients have improved markedly in recent years, much remains to be done to make antemortem diagnostics in other mammals accurate and practical enough for routine applications in agriculture and wildlife. Further study is also required to understand the extent to which prionlike mechanisms contribute to the etiology and pathogenesis of the many proteinopathies that involve the abnormal assembly of proteins other than PrP. In assessing the clinical significance of prionlike characteristics of various protein aggregates, a key issue is not only transmissibility but also pathogenicity; that is, do replicating and spreading aggregates necessarily cause disease in the new host75,93? This issue is analogous to the distinction between virulent vs nonvirulent viral or bacterial infections. When transmitting TSE prions from one species to another, or when inoculating synthetic PrP aggregates, the inocula can sometimes initiate extensive conversion of the host’s endogenous PrP molecules without causing clinical disease or, even TSE-associated neuropathological lesions, within the lifespan of the host.32,75,163,213,246,322 Indeed, different 767

wild-type hamster PrP amyloid fibrils have been shown to range in lethality across many orders of magnitude and have varying abilities to replicate and/or cause neuropathology (Fig. 22.15). Also, some neuropathogenic mutant forms of PrP can accumulate in the host without being transmissible to other hosts.93

FIGURE 22.15 Range of transmissibility and pathogenicity of hamster PrP amyloid fibrils. Although all of the depicted amyloid fibrils are capable of continuous propagation either in vivo or in vitro, they differ dramatically in their infectious properties when inoculated intracerebrally into hamsters. Negatively stained transmission electron micrographs show fibrillar ultrastructures (bar = 100 nm). Although the fibrils depicted in the second panel from left were prepared with detergent and only barely lethal,195 it is important to note that other synthetic recombinant prions prepared with cofactors can be orders of magnitude more lethal per unit protein (lethal unit of ~10−10 g).400

(Adapted from Figure 1 of Caughey B, Kraus A. Transmissibility versus pathogenicity of self-propagating protein aggregates. Viruses. 2019;11(11):1044. http://creativecommons.org/licenses/by/4.0/.) In humans, as noted above, concerns remain about the implications of finding abnormal PrP deposits in the appendices of 1 out of every 2000 Britons in an age cohort that could have been exposed to BSE prions. Related concerns about the potential transmissibility of Aβ amyloid have been raised recently based on the detection of Aβ pathology in patients with early-onset CAA or who had undergone prior neurosurgeries,184 dura mater grafts,168 or injections of cadaveric growth hormone.183 However, an extensive analysis of a cohort of people in the latter category provided evidence that exposures to injected Aβ or α-synuclein amyloids were not epidemiologically significant causes of neurological disease.18,181 These examples emphasize the epidemiological importance of considering not only prionlike spreading potential but also the clinical manifestations, if any, of accumulating self-propagating protein assemblies.

Acknowledgments This work was supported in part by the Intramural Research Program of the NIAID. We thank Drs. Suzette Priola, Cathryn Haigh, Ankit Srivastava, Moses Leavens, James Carroll and Efrosini Artikis, and Kachi Isiofia for critiquing this manuscript. We thank James Striebel and Bruce Chesebro for assistance with Figures 22.10 and 22.11.

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Index Note: Page number followed by “f” and “t” indicates figure and table respectively. A Abacavir, 373 Ab-dependent cell-mediated cytotoxicity (ADCC) mechanisms, 411 Aberrant growth control, 28 Abortive infection, 192 Acanthamoeba polyphaga mimivirus (APMV), 467, 473–474 Acetyl-CoA, 176–178 Acquired human prion diseases, 640–641 iatrogenic cases, 640–641 Acquired immunodeficiency syndrome (AIDS), 12, 334, 403 Activated antiviral T lymphocytes, 249–250 Active surveillance, 334 Acute infection, 192–193, 193f vs chronic, host response to, 193–194 Acyclovir (ACV), 353–354, 366f–367f, 367 mechanisms of action of, 367 Acylglycerol lipids, 181–182 Adamantane derivatives, 360–361, 361f Adaptive immune memory, 276–278 B-cell memory long-lived plasma cells, 277 long-term humoral immunity, 277–278 memory B cells, 276–277 T-cell memory, 278 viral replication, 120 Adaptive immune system, 214, 216–217 adaptive immune memory, 276–278 antigen-presenting cells and induction, 258–261 dendritic cells, T lymphocytes in secondary lymphoid organs, 258–259 T and B lymphocytes in secondary lymphoid organs, 259–261 cell types, 248, 248t circulating molecules, 249 development and architecture CD1 family of class Ib genes and lipid recognition, 256–257 genes and molecules, 255–256 secondary lymphoid organs, structure and function, 253–255 T and B lymphocytes, 250–253, 251f, 253t 784

T-cell receptor, 257 effector activities of B cells, 269–271 of T cells, 273–276 nonhematopoietic body cells, 249 primary host defenses, 248 viral antigen recognition B cells, 262–265 by T cells, 265–269 viral evasion of cellular (T-cell) immunity, 279–281 of humoral (B-cell) immunity, 279, 279t viral strategies, 278, 279t virus-induced immune dysregulation and autoimmune disease, 281–282 Adeno-associated virus vectors, 431 Adenoviridae, 126f Adenovirus capsid structure, 60f, 61–62 vaccines, 419 vectors, 430–431 Adjuvants, 433–434 Adnaviria, 23, 117 Aerobic glycolysis, 175–176 Afferent lymphatic vessels, 254 Agglutination assays, serology, nucleic acid detection, 460 AIDS, 12, 334, 403 AIM2-ASC inflammasome, 231–232 Alfalfa mosaic virus (AMV), 69 Allosteric controls, 175 Allosteric regulation of glycolysis, 174–175 Alphaviral MTase-GTase, 323 Alphavirus vectors, 431–432 Alum, 433 α-ketoglutarate, 178 Amantadine, 353–354, 360–361, 361f Amber mutations, 8 Amino acids, 184 Amplicon, 453 Analyte-specific reagent (ASR), 457 Animal models, 201–204 785

human viruses, 202 natural infection models, 201–202 physiology of, 204 selection of, 202–204, 203f Animal prion diseases bovine spongiform encephalopathy, 644–645 camel prion disease, 645 chronic wasting disease (CWD), 645–646 mammalian prion diseases, 645–646 scrapie in small ruminants, 645 Animal viruses antivirals, 11–12 cell culture technology and discovery, 9–10 emerging viruses, 12–13 eukaryotic gene regulation, 10 molecular and cell biology era, 10 oncology, 11 recombinant DNA revolution, 10–11 Antemortem diagnosis, 646 Antibodies active against virus-infected cells, 411 Antibodies in vivo, antiviral activity of, 412 Antibody escape mechanisms, 411 Antibody response, kinetics, 459 Antibody-based methods, 463 Antibody-dependent enhancement (ADE), 421–422 Antibody-mediated virus neutralization isotype, mechanisms of, 409 mechanisms of, 407–408 Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), 530–531 Anti-CRISPR proteins (ACRs), 325–326 Antidefense systems, 326 Antigen detection diagnostic virology, 451–452 enzyme immunoassay, 452 fluorescent antibody staining, 451–452 membrane immunoassay, 452, 452f Antigenic peptide generation, 280 Anti-HBV nucleoside analogues, 365, 366f–367f, 371–374 Antiherpesvirus, 365, 366f–367f nucleoside analogs, 370–371 786

cidofovir and brincidofovir, 366f–367f, 370 nucleoside analog, 370–371 penciclovir and famciclovir, 366f–367f, 370 Anti-HIV nonnucleoside reverse transcriptase inhibitors, 375, 376f Anti-HIV protease inhibitors, 381f Antipoxvirus nucleoside analogues, 365, 366f–367f Antiprion systems in Saccharomyces cerevisiae, 582–583, 582f Btn2 and Cur1 cure [URE3] isolates, 582–583 Hsp104 disaggregase, antiprion activity of, 583 inositol polyphosphates and prion propagation, 583 inter- or intraspecies barrier to prion transmission, 582 Lug1 lets [URE3] grow, 583 nonsense-mediated decay components Upf1,2,3, 583 ribosome-associated chaperones, 583 Sis1 blocks [PSI+] lethality, 583 Antirepressor, 604 Anti-RNA virus nucleoside analogues, 365, 366f–367f, 374–375 remdesivir, 366f–367f, 374–375 ribavirin, 374 sofosbuvir, 366f–367f, 374 Antisigma, 598 Antiviral adaptive immune responses, regulatory T-cell function, 275–276 Antiviral agents and barriers, medical value of, 353 drug development, process of, 353–354 drug mechanisms and resistance, 354–356 antiviral drug targets, aspects of, 355 drug selectivity, resistance, and drug targets, 354–355 factors, 355–356 basic science, 354 mechanisms of, 356–385 adamantane derivatives, 360–361, 361f anti-herpesvirus nucleoside analogs, 370–371 anti-HIV and HBV nucleoside analogs, 371–374 anti-RNA virus nucleoside analogs, 374–375 antiviral therapies, 385 hepatitis C virus NS5A protein, inhibitors of, 377 HIV integration, inhibitors of, 377–379 nonnucleoside inhibitors of polymerases, 375 nucleoside analogs targeting polymerases, 365–370 787

SARSCoV-2 Mpro inhibitor, 365 targeting drugs to, 356, 357f–358f viral assembly/egress, inhibition of, 379–385 viral entry, inhibition of, 356–360 viral gene expression, inhibition of, 361–365 viral genome replication, inhibition of, 365, 366f–367f viral genome replication, prospects for new inhibitors and targets, 379 principles of, 385–390 drug resistance, 385–386 herpesviruses therapy, 387–388 HIV therapy, 386–387, 387f poxvirus therapy, 389–390 SARS-CoV-2 therapy, 389 viral hepatitis therapy, 388–389 Antiviral defense recruited cells, 240–241 tissue-resident innate immune cells, 238–240 Antiviral drugs for basic science, 354 medical value of, 353 Antiviral factors, 195 Antiviral IgA Abs function, 413 Antiviral ISG effectors, 236 Antiviral RNAi, 512–513 in plant virus infection, 512–513 secondary sRNA production in, 513 Antiviral susceptibility testing, 461–463 genotypic assays, 462–463 phenotypic assays, 461–462, 462t Antiviral therapies, 385 Apoptosis, 539–542, 540f Baculoviruses-induced, 539–542, 540f apoptotic suppressors, 541 signaling of, 541–542 Arbovirus infections, 338 Arenaviridae, 24 Arg-Gly-Asp (RGD) sequence, 99 Artificial intelligence (AI) program, 418 Artverviricota, 24 AS04 (Adjuvant System 04), 433 788

AS01B, 434

Ascoviridae, 546–547, 546f classification and structure of, 546 disease, 547 host range, 547 replications, 546–547 transmission, 547 Assembly units, 55–56, 59 Atachment site, 9 Attack rate, 333–334 Attenuation of live viruses, genetic basis, 420–421 Autographa californica nucleopolyhedrovirus (AcMNPV), 529, 531–535 very late occlusion stage of, 538f Autophagy and plant virus infection, 514 Axonal rapid endosomal sorting and transport-dependent aggregation (ARESTA), 632–633 B B cells effector activities antiviral activities of antibody in vitro, 269–271 antiviral activities of antibody in vivo, 271–273 viral antigen recognition antigenicity and immunogenicity, 262–263 B-cell activation by viral antigens, 264–265 B-cell epitopes, 262 primary B-cell repertoire generation, 263–264 Baby hamster kidney (BHK) cells, 432 Bacillus anthracis, 1–4 Bacterial vectors, 430 Bacteriophages, 590 antagonistic interactions, 618 Bacillus megaterium phage G, 605 Caudovirales, dsDNA tailed phages, 591–601 genome sizes, 591 head of, 591–592 λ phages, 592 lambdoid phages, 592–596 T4 bacteriophage (See T4 bacteriophage) temperate phages, 591–592 virulent phages, 591–592 communities and ecology, 619 789

Corticoviridae, 612–613 Cystoviridae, 612, 614 DNA replication, 617–618 cellular machinery, 618 transcription, 617 translation, 617–618 encapsulins, 615 evolution of, 613–614, 615f common ancestry of archaeal and eukaryal viruses with phages, 614 genome mosaicism, 613–614 gene transfer agents (GTAs), 615 history of, 590–591 HK97, 601–602, 603f host interactions, 617 Inoviridae, 609–611 Leviviridae, 611–612 Microviridae, 609, 609f Mu, 605–606 ø29, 606–607, 607f P22, 603–604 P1 and N15, 606 P2 and P4, 604–605 parasitic interactions, 617 phages and the birth of molecular biology,1938–1970, 7 Plasmaviridae, 613 PRD1, 63f prototypes and characteristics, 591t pyocins, 614–615 symbiotic interactions, 618–619 T7, 602–603 Tectiviridae, 607–608 therapeutic uses of, 619–620 type VI secretion systems (T6SS), 615–617, 616f Baculoviridae, 531–542. See also Baculoviruses classification of, 531 Baculoviruses, 530–531 alterations of host, 539 apoptosis, 539–542, 540f apoptotic suppressors, 541 signaling of, 541–542 790

budded virus (BV), 531–532, 532f disease progression in insects, 539 fates of baculovirus-infected cells, 535f few polyhedra (FP) phenotype, 542 gene expression, 533–534 genome structure, 532–533 host transposons, 542 occluded virus (OV), 531–532, 532f replication stages, 533–539, 534f attachment, endocytosis, and uncoating, 533 DNA replication and late expression factors, 537t early phase, 533–535 late phase, 535–538 primary and secondary infection, 533–539 very late phase, 538, 538f virus assembly, budding, and occlusion, 538–539 structure, 531–532 transactivator IE1 structure, 536f vectors, 529–530 Baculovirus-produced influenza hemagglutinins, 425 Baloxavir acid (BXA), 362, 363f viral gene expression, inhibition of, 362, 363f Baloxavir marboxil (BXM), 362, 363f Baltimore classes and monophyletic realms of viruses, 313–314, 313f Bamford viruses, hallmark proteins, 469, 469f Barley yellow dwarf virus (BYDV), 506–507 B-cell activation, 261 B-cell memory long-lived plasma cells, 277 long-term humoral immunity, 277–278 memory B cells, 276–277 Beijerinck, Martinus, 5 Bilayer fusion, 78, 78f BILN-2061, 363–365 Binary movement block (BMB), 509–510 Binding assays, 460 Biojector, 435 Biopesticide viruses for insect control, 530–531 Bizarre bovine papillomavirus (BPV) E5 protein, 296 Bluetongue virus (BTV), 107 791

Boceprevir, 362–363, 364f Bombyx mori nucleopolyhedrovirus (BmNPV), 529–530 Bovine amyloidotic spongiform encephalopathy (BASE), 642 Bovine papillomavirus (BPV), cryo-EM, 51, 51f–53f Bovine rotavirus vaccines, 428 Bovine spongiform encephalopathy, 641–642, 644–645 Bracoviruses, 544 Brincidofovir, 366f–367f, 370, 389–390 Broadly protective vaccines, 436 Bronchoalveolar lavage (BAL) fluids, 412–413 Budded virus (BV), 531–533, 532f Bulevirtide, 356–357 Bunyaviridae, 24 Burkitt lymphomas, 11 Burst size, 34 C Cafeteria roenbergensis virus (CroV), 473–474 Calicivirus-receptor complex at low pH, 87f Calomys callosus, 350 Camel prion disease, 645 Campoletis sonorensis polydnavirus (CsIV), 544 Cancer cell transformation, 287 as disease, 286–287 fraction of, by tumor viruses, 298, 299f metabolism, using both cell and animal models, 170 sequencing data, 306 Candida albicans plasmid, retrotransposon and line elements, 576 Canine parvovirus (CPV), 56f Cap-dependent endonuclease (CEN), 362 Cap-independent translation, 504–506, 506f enhancers, 506–507 Capping enzymes, viruses, 322–323, 323f Capsid, 50 Capsomeres, 55–56 Carbohydrates, 99–100 Cardiolipins (CLs), 182 Case fatality ratio, 339 Case infection, 339 ratio, 339 792

Case-control studies, 336–337, 336t Catabolism, 170 Caudovirales, dsDNA tailed phages, 591–601 genome sizes, 591 head of, 591–592 λ phages, 592 lambdoid phages, 592–596 T4 bacteriophage (See T4 bacteriophage) temperate phages, 591–592 virulent phages, 591–592 Caveolar and lipid raft-mediated endocytosis, variants, 102 C-capsid, 379 CCR5, 357, 360 CD4, 357 CD8+ effector mechanisms, 273–274 CD1 family of class Ib genes and lipid recognition, 256–257 CD4 T cells, 404–405, 414–415 CD8 T cells, 404–405, 413–414 CD8+ T-cell exhaustion/dysfunction, 275 CD4+ TE effector mechanisms, 274–275 CDR3 loops, of antibody variable region, 409 Cedratvirus, 484–487 genome structure and organization, 486 stages of replication, 486–487 virion structure, 484–486, 485f Cedratvirus getuliensis, 486–487 Cell, metabolic program of, 170–171 Cell culture, 26, 199 advantages and disadvantages, 28–29 animal cells in vitro, 26 cell lines, 28 cell strains, 27, 27f organ culture, 26 organoid cultures, 26, 29, 29f primary cell cultures, 27 primary explant culture, 26 subcultivation, 27 transformation, 28 in vivo, 26 Cell lines, 28 793

Cell lysis, 611 Cell strains, 27, 27f Cell transformation model of cancer, 287 and tumor formation by EBV, 297 Cell-associated and noncanonical spread of infection, 164–165 Cell-autonomous virus recognition, 228–232 NOD-like receptors, 230–232, 230f RIG-I–like receptors, 228–230, 229f Cell-based carriers, 435 Cellular destruction, 9 Cellular entry, of animal viruses attachment factors, 96–97 barrier, 96 carbohydrates, role of, 99–100 caveolar and lipid raft-mediated endocytosis, 102 clathrin-mediated endocytosis, 101f, 102, 103f direct cell-to-cell transmission, 110–111, 111f endocytic pathway, 104–105, 104f–105f endocytic pathways of infection, 100–102, 101f intracellular trafficking, 107–108 macropinocytosis and macropinocytosis-like mechanisms, 102–104 mobility of cell-associated viruses, 100 nuclear import, 109–110, 109f penetration by membrane fusion, 105–106, 106f nonenveloped viruses, 106–107 perspectives, 111–112 virus receptors, 96–97 virus-induced signals, 100 Cellular membranes, enveloped viruses at endoplasmic reticulum, 151–153 at endoplasmic reticulum-golgi intermediate compartment, 153 in golgi complex, 153–154 at plasma membrane, 154–155 viral glycoproteins in polarized epithelial cells, 156 Cellular protooncogenes, insertional activation, 294–295 Cellular responses, 405 Central nervous system neurons, 412 Cerebral amyloid angiopathy (CAA), 640 794

Cerebrospinal fluid analysis, 643–644 Cerebrospinal fluid serology, 461 Cervical intraepithelial neoplasia (CIN) grade 2, 423 cGAS virus recognition pathway, 229–230, 230f cGAS-STING signaling pathways, 230 Chamberland, Charles, 4 Chamberland filter, 5, 5f Chaperones involvement in prion propagation, 581–582, 581f ribosome-associated, 583 Chikungunya virus (CHIKV), 194 Chimeric live virus reassortant and recombinant vaccines, 428–429 Chimeric recombinant virus vectors, 427–429 Chinese hamster ovary (CHO) cells, 425 Chlorella, 467–468 Chloroviruses, 472 Cholesterol, 182–184, 183f metabolism, 185 Cholesterol recognition/interaction amino acid consensus (CRAC), 507–509 Chronic infection, 192–193, 193f vs acute, host response to, 193–194 serology in, 459 Chronic wasting disease (CWD), 624, 642, 645–646 Cidofovir, 366f–367f, 370, 388 Ciluprevir, 363–365, 364f Circular genetic map, T-even phages, 8 Circulating vaccine-derived poliovirus type 2 (cVDPV2), 403 Citrate, 176–178 Class I fusion proteins, virus entry, structure biology of, 78–83 HIV-1 Env, 81, 82f influenza A virus HA, 78–81, 79f, 80f, 81f paramyxovirus and pneumovirus F, 81–83, 82f Clathrin-mediated endocytic pathway, 101f, 102, 103f Clinical Laboratory Improvement Amendments (CLIAs), 452 Clinical virus disease research, 199 Closteroviridae, 512 Cluster randomized trials, 336 Co-delivery vaccines, 435 Codon pair deoptimization, 420–421 Cohort studies, 335–336, 335f 795

Community (herd) immunity, 400–403 Complementary DNA (cDNA), 567 Conjugates, 435 Connector, in bacteriophages, 591–592 Contagious living fluid, 4 Contamination, nucleic acid amplification assays, 457 Continuous cell lines, 28 Copy number control (CNC), 560–561, 574–575 of 20S RNA and 23S RNA, 570 Coreceptors, 97, 358–360, 359f Correlates of protection (CoPs), 404–405 Cortical actin network, 96 Cortical ribboning restricted diffusion, 642–643 Corticoviridae, 612–613 Cotesia congregata bracovirus (CcBV), 544 COVID-19 pandemic, 15–16, 101–102, 247–248 Coxsackie and adenovirus receptor (CAR), 97, 98t–99t CpG 1018 adjuvant, 434 Creutzfeldt-Jakob disease (CJD), 624, 636 cerebrospinal fluid analysis for, 643–644 electroencephalography for, 643 imaging, 642–643 non-PRNP single nucleotide polymorphisms, 636–637 PRNP gene polymorphisms, 636, 639f susceptibility and phenotype, 636–637 Crisis, 27 CRISPR effector complexes, 325–326 CRISPR gene editing, 203–204 CRISPR system, 618 CRISPR-based strategies, 35 Cryogenic electron microscopy (cryo-EM) bovine papillomavirus, 51, 51f–53f limitation, 51 Cryogenic electron tomography (cryo-ET) herpes simplex virus type 1, 51, 52f immature and mature HIV-1 particles, 51, 53f Cryphonectria parasitica, 568 coding information and protein processing of, 569f infectious cDNA clones and biological control, 570 mitochondrial RNA replicon NB631, 570 796

RNAi in, 568–569 Cryphonectria, reovirus of, 567–568 CTXɸ phage, 611 C-type lectin receptors (CLRs), 228, 233–234 Cultured cell types, 27f Cydia pomonella granulovirus (CpGV), 531 Cystoviridae, 612, 614 Cytochrome oxidase subunit I (COX1) gene, 571 Cytokines, 432–433 and cytokine receptor homologs, 281 Cytolytic viruses, 194 Cytomegaloviruses, 179 Cytopathic effects, 25–26, 29 Cytoplasmic targeting/retention signal (CTRS), 149–150 Cytotoxic T lymphocytes (CTLs), 12, 412 D Dane particle, 422–423 Danger-associated molecular patterns (DAMPs), 515 Dead end, 342 Decoration proteins, 600, 602 Deep mutational scanning, 46 Defective interfering (DI) particles, 46 Deglycosylated protein Ags, 409–410 Delbrück “type” phages, 596 Delivery vehicles, 434–435 cell-based carriers, 435 conjugates, 435 lipid-based carriers, 434 mechanical devices, 435 synthetic particles, 434–435 Dendritic cells (DCs), 228, 240, 240f, 435 activation/maturation, 259 direct infection, 259 inflammatory mediators, 259 T lymphocytes in secondary lymphoid organs, 258–259 uptake of virus, 259 Dengue virus (DENV), 408–411 replication, 180 DENV infection, 214 DENV-specific afucosylated IgG1, 214 797

Descriptive epidemiology, 337–338 DHAP (dihydroxyacetone phosphate), 173 Diagnostic virology, 448 categories of testing, 449t cytology and histology, 451–452, 451f antigen detection, 451–452 history of, 448, 449t methods used in, 449–451 electron microscopy, 450–451 light microscopy, 451 viral culture, 449–450, 450t, 450f multiple test methods, 448 nucleic acid detection, 453–463 antiviral susceptibility testing, 461–463 nucleic acid amplification assays, 453–458 serology, 458–461, 459t perspective, 465 selected clinical problems, 463 specimens for, 448 virus discovery and metagenomics, 463–465 prospects and challenges for future, 464–465 representative novel human viruses discovered, 464 Dicistroviridae, 529, 552–553 Dicistrovirus, 552–554 genome organization and novel translation initiation, 553–554 prevalence and transmission, 554 Diffusion tensor imaging (DTI), 643 Diffusion-weighted imaging (DWI), 642–643 Dimeric IgA, 412 Direct virus-induced damage, 194 Disease-associated viruses molecular biology, 192 molecular pathogenesis, 192 DNA vaccines, 427 DNA viral genomes vs RNA genomes, 122 viral replication strategies expression and replication, 125–128, 125f–128f latent and persistent infections, 130 mechanisms of, 129–130 798

regulation of, 128–129 transcription, 129–130 viral oncogenes and neoplastic transformation, 130–131 DNA virus oncoproteins, 296t Herpesvirus oncoproteins, 297–298 inactivation, tumor suppressor pathways, 296–297 mitogenic signaling pathways, activation, 296 DNA viruses, 159–160 DNA-dependent DNA polymerase (DdDP), 314–315 DNA-dependent RNA polymerases (RNAP) and transcription factors, 321–322 DNA-RNA hybrid, 454 Docosahexaenoic acid, 180–181 Double jelly roll capsid proteins, 317 Double mutants and siblings, 38 Double stranded DNA (dsDNA) binding, 229–230 Double-Psi Beta Barrel (DPBB) domains, 321–322 Double-stranded RNA viruses, 561–568 mycoreovirus facilitates infection by preventing heterokaryon incompatibility, 568 Partitiviridae, 567 reovirus of Cryphonectria, 567–568 S. cerevisiae, Totivirus type species L-A of, 561–567 antiviral systems, 566 cap-snatching by Gag separates strands, 565–566 killer phenomenon of, 562f L-A genome structure, 562–564 L-A virion structure, 561–562, 562f N-acetylation of Gag protein by mak3p, 567 Nuc1 and Ski3 block viral lethality in meiosis, 566 positive-strand synthesis, 564–565 posttranslational modification, 566–567 replication (negative-strand synthesis), 567 ribosomal frameshifting, 565 60S subunits, 565 Ski2 antiviral system, 566 stages in replication cycle, 564, 564f transcription reaction, 564–565 translation, 565–566 viral assembly, 567 Drosophila melanogaster, 540, 550 799

Drought tolerance in plants, 519 Drug resistance, 385–386 clinical impact of, 386 strategies to, 386 Drug selectivity, 354–355 Drug targets, 354–355 Drug-resistant herpesviruses, 386 DS-Cav1, 410–411 Duplodnaviria, 23, 117 E E coli, 600 DnaK and DnaJ proteins, 618 ɸX174 phage, 609 HK97 phage, 614 K12 strain of, 592 T7 phage, 602 Eastern equine encephalitis virus (EEEV), 194 Ebola glycoprotein (ZMapp), 407 Ebola virus disease (EVD), 407 Eclipse, 34, 106–107 Effector molecules, 174 Effector-triggered immunity (ETI), 515 Efficient antibody interactions, antigens critical, 409–410 Eicosapentaenoic acid, 180–181 Electroencephalography (EEG), 643 Electron microscopy, diagnostic virology, 450–451 Electron paramagnetic resonance (EPR), 628 Electron-lucent viral factory, 479 Elimination, 403–404 ELISA, 200, 646 Elongated shells, with icosahedral caps, 64, 65f Emergent viruses, 345–346 Empty viruslike particles (eVLPs), 520 Emtricitabine, 373 Encapsulins, 615 Encephalitic viruses, 205–206 Endocytosis caveolar and lipid raft–mediated endocytosis, 102 clathrin-mediated endocytic pathway, 101f, 102, 103f electron microscopy of virus, 102, 103f 800

mechanisms, 100–102, 101f Endogenous viral elements (EVFs), 496 Endoplasmic reticulum, enveloped viruses, 151–153 Endoplasmic reticulum-golgi intermediate compartment, 153 Endosomes pathway, 104, 104f–105f viruses entry, 104, 105f Endpoint method, 32 Enfuvirtide, 360 Envelope fusion protein (EFP), 531–532 Enveloped viruses budding of, 72f cellular membranes at endoplasmic reticulum, 151–153 at endoplasmic reticulum-golgi intermediate compartment, 153 in golgi complex, 153–154 at plasma membrane, 154–155 viral glycoproteins in polarized epithelial cells, 156 in nucleus, 148–149 Enveloped viruslike particles, 424, 424f Enzyme immunoassay (EIA), antigen detection, diagnostic virology, 452 Enzyme-based genome editing methods, 201 Epidemics, 344–346 causes of, 344–346 emergent or re-emergent viruses, 345–346, 345f population susceptibility, increase in, 345 susceptible population, increase in, 345 viral virulence, increase in, 344–345 investigation, 346 common source epidemic, 346 point source outbreak, 346 propagated epidemic, 346 Epidemiology, 333 applications of, 348–350 control measures, development and assessment of, 350, 350t vaccine efficacy and safety, evaluation of, 349–350, 350t definitions and methods of, 333–337 case-control studies, 336–337, 336t cohort studies, 335–336, 335f disease burden, data sources for, 334–335 801

measures of disease burden, 333–334, 334f descriptive epidemiology, 337–338 person, 337 place, 337–338 time, 338 epidemics, 344–346 causes of, 344–346 infectious disease epidemiology, concepts of, 338–340 case infection and fatality ratios, 339 generation time and serial interva, 340 incubation, latent and infectious periods, 339–340 parameters, 338–339 proportion infectious, 339 proportion susceptible, 338–339 rate of contact, 339 susceptibility and immunity, 338 transmission of viruses, 341–344, 341t endemic and epidemic patterns of, 342 within host persistence, 342 modeling viral dynamics, 343 multiple host species, viruses maintained in, 341–342, 341t phylodynamics, 343–344 quantifying transmission and basic reproductive ratio, 342–343 single host species, viruses maintained in, 341 terminal hosts, 342 viral emergence, 344, 344f viruses, perpetuation and eradication of, 346–348 eradication, requirements for, 347–348 large populations, 347, 347t small populations, 347 Epstein-Barr virus, 11, 303 Equine infectious anemia virus (EIAV), 421–422 Eradication, 346, 403–404 requirements for, 347–348 measles virus, 348 polioviruses, 348 rinderpest virus, 348 ER-Golgi intermediate compartment (ERGIC), 229–230 Error catastrophe, 369–370 Escherichia coli–replicating bacmid technology, 535–536 802

Eukaryotic cells, production of viral proteins, 425 Eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3), 185 Euploid, 27 Evasion strategy mechanisms, 241–242 of DNA vs RNA viruses, 242–243 Evolution, viral proteins dark matter of virus proteomes, 326–327 knowable unknowns, 326–327 unknowable unknowns, 326–327 exaptation and recruitment of host proteins, 327–328, 327f functional gene categories in viral genomes, 328, 328f gene exchange, hosts, 312 lineage-specific virus proteins, core processes, 323–324 moderately conserved proteins, virus functions capping enzymes, 322–323, 323f DNA-dependent RNA polymerases and transcription factors, 321–322 P-loop enzymes, 319–320 proteases, 320–321 silencing suppressors, 326–327 virus hallmark proteins family B DNA polymerases, 314–315 helicases and genome packaging NTPases, 316–317 and implications, origin, 318–319 palm/RRM domain, virus replication machinery, 316 reverse transcriptase, 314 RNA-dependent RNA polymerase, 314 rolling circle replication initiation endonuclease, 315, 315f virus capsid proteins, 317 virus proteins, virus-host interactions, 324–326 Ex vivo systems, 191 Exosomes, 434 Extended Asfarviridae, 482–484 genome structure and organization, 483–484 stages of replication, 483f, 484 virion structure, 482–483, 483f F Famciclovir, 366f–367f, 370 Family B DNA polymerases, 314–315 Fatal familial insomnia (FFI), 638–639 Fatality ratios, 339 803

Fatty acids (FA) desaturation, 180–181 elongation, 180–181 synthesis, 180–181 Faustovirus, 482–484 Fibril acquisition area, 476 Fibrillarin, 512 Fibritin, 600 FilmArray Respiratory Panel, 458 Filterable viruses, 4 Flasuviricetes, 23 Flaviviridae, 23 Flavivirus E protein, 83, 84f Flavivirus genome replication, 151–153, 152f Flavivirus structure, 61f Fluorescence resonance energy transfer (FRET), 453 Fluorescent antibody staining, antigen detection, diagnostic virology, 451–452 Fluorescent cell units, 35 Force of infection, 334 Formalin-inactivated RSV (FI-RSV) vaccine, 421 Forward genetics, 36 Forward glycolytic reactions, 171 Foscarnet, 375, 376f, 388 Fostemsavir, 357–358, 359f FtsK-like genome packaging ATPase, 317 Fumarate, 178 Functional assays, serology, nucleic acid detection, 460 Functional genomics technologies, 200–201 FUS3 MAP kinase, mutation in, 575 Fusion inhibition (FI), 407–408 Fusion pore, 78 G G bacteriophages, 605 G protein signaling, 569 Gag precursor proteins, 149–150 N-acetylation, by mak3p, 567 Gag-Gag interactions, 162–163 Gag-Pol fusion protein, 561, 565 Gain-of-function mutations in STING, 230 Ganciclovir (GCV), 366f–367f, 367–369 804

mechanisms of action, 367 Gardasil, 433 Gastric carcinoma, 303 Geminiviruses, 502, 511 infection cycle and interactions with host cell, 503f Gene delivery, 530 vaccines by, 426–433 DNA vaccines, 427 gene-based vectors, limitations of, 432 mRNA vaccines, 427 nucleic acid vaccines, 426–427 replication-competent vectors, 427–430 replication-defective vectors, 430–432 viral vectors as vaccines, 432–433 Gene exchange, hosts, 312 Gene gangs, 471–472 Gene therapy, 530 Gene transduction, 530 Gene transfer, 287 Gene transfer agents (GTAs), 615 Gene-based vectors, limitations of, 432 Generalized transduction, 603–604 Generation time, 340 Genetic engineering, 520 Genetic prion diseases, 638–640 implications of, 640 PrP amyloidosis, 639–640, 639f thalamic degeneration, 638–639 Genome replication and expression, 319 Genome uncoating, 108 Genome wide association studies (GWAS), 209 Genomic packaging, 161 Genomic sequencing, 344 Genomic technologies, 206 Genotypic assays, 462–463 resistance assays, 457 Germ theory of disease, 1 Germline transmission, 341 Gerstmann-Sträussler-Scheinker syndrome (GSS), 636, 638–640 Giant viruses, 312 805

classification and evolutionary relationships, 469–480 discovery and characterization, 467 extended Asfarviridae, 482–484 genome structure and organization, 483–484 stages of replication, 483f, 484 virion structure, 482–483, 483f genomic and structural features of, 467, 468f history, 467–468 medusavirus, 489–490 genome structure and organization, 489 stages of replication, 489–490, 490f virion structure, 489 Mimiviridae, 473–477 genome structure and organization, 474–476 stages of replication, 479–480 virion structure, 474, 475f mollivirus, 487–489 genome structure and organization, 487–488 stages of replication, 488–489 virion structure, 487, 488f pandoraviruses, 480–482 genome structure and organization, 481–482 stages of replication, 480f, 482 virion structure, 480–481, 480f Phycodnaviridae, 470 genome structure and organization, 470–472 stages of replication, 472–473 virion structure, 470, 471f pithovirus, cedratvirus and orpheovirus, 484–487 genome structure and organization, 486 stages of replication, 486–487 virion structure, 484–486, 485f Glassy transformation, 6 Glecaprevir, 364f Global Outbreak Alert and Response Network (GOARN), 334 Gluconeogenesis, 171 Glutaminolysis, 178 Glycolysis allosteric regulation of, 174–175 biosynthetic pathways, 171–173, 172f 806

controls of, 174 glucose transporters, 174 growth factors and receptors, 174 metabolic profiles, 173–174 pro-growth nutrient environment, 174 signaling pathway activation, 174 Glycophosphatidylinositol (GPI) anchor, 630, 636 Glycoprotein-derived peptide, 356–357 Gly-3-P (glyceraldehyde-3-phosphate), 173 Golgi complex, 147 enveloped viruses, 153–154 G-6-P (glucose-6-phosphate), 173 Grazoprevir, 364f GRP78, 479–480 GTPase activating proteins (GAPs), 294 Guillain-Barré syndrome (GBS), 422 H Hadakaviruses, 570–571 Hamster-mouse transmission barrier, 631f Hantavirus pulmonary syndrome (HPS), 349 HB surface antigen (sAg) vaccine, 403 HBV nucleoside analogs, 371–374 HBV therapy, 388 HCMV protein kinase, 370–371 HCMV therapies, 388 HCMV-infected cells, 371 HCV NS3/4A protease inhibitors, 362–365 HCV therapy, 388–389 Headful packaging process, 599 Heavy-chain CDR3 (HCDR3), 409 Helical symmetry, 52–54, 54f Helicases and genome packaging NTPases, 316–317, 316f FtsK-like genome packaging ATPase, 317 large terminase subunit, 316–317 superfamily 3 helicases, 316 Helicoverpa armigera stunt virus (HaSV), 552 Heliothis virescens ascovirus (HvAV), 547 Helminthosporium victoriae, 565 Hemadsorption, 29–30 hemagglutination inhibition (HAI) titer, 404–405 807

Hemifusion stalk, 78 Hepadnaviridae, 128f Hepatitis B virus (HBV), 304, 339–340, 371–374 drug resistance alterations, locations of, 371, 372f NPC, 110, 110f vaccination, 304, 304f Hepatitis C virus (HCV), 153, 305, 353 inhibitors of, 376f, 377 Herpes simplex virus 1 (HSV-1), 109f cerebral organoids, 29 therapy, 387 Herpesviridae, 22, 24, 127f Herpesviruses, 387–388 envelopment in cytoplasm, 156–157, 157f HCMV therapies, 388 HSV and VZV therapies, 387 oncoproteins, 297–298 vectors, 429, 432 Hershey-Chase experiment, 8 Heterokaryon incompatibility systems, 579f mycoreovirus facilitates infection by preventing, 568 Heterologous immunity, 214–215 HET-s prion domain, 580 Hexokinase, 175 High throughput analysis of host gene function, 201 High throughput genetic screening, 206 High throughput, whole genome sequencing methodologies, 207 High-throughput sequencing technology, 457 impact of advances in, 436–437 Histidine triad nucleotide-binding protein1 (Hint1), 374 HIV-induced acquired immunodeficiency syndrome (AIDS), 214 HK97, 317 bacteriophages, 601–602, 603f fold, 67 Home-brew assays, 457–458 Homologous recombination, 613 Host antigens, inadvertent induction of antibodies, 411–412 Host immune system controlling viral infection, 212–217, 213f innate system, 217 808

Host kinases, nonchain terminating analog phosphorylated, 366f–367f, 369–370 Host-defense mechanisms, viral replication, 120 Hsp70s, in prion propagation, 581 Hsp90s, in prion propagation, 581–582 Human autoimmunity, 217 Human endogenous retroviruses (HERVs), 17, 306 Human immunodeficiency virus (HIV) infection, 305, 334, 353, 403, 407 by classes of antiviral drugs, 356, 358f HIV Env, 82f HIV gp41–mediated fusion model, 357–358, 359f HIV integrase, inhibitors of, 377–379, 378f inhibitors of, 377–379 HIV integrase, inhibitors of, 377–379, 378f lenacapavir, 378f, 379 protease inhibitors, 382 therapy, 386–387, 387f type 1 (HIV-1), 24, 76–77, 77f, 97 drug resistance alterations, locations of, 371, 372f capsids, NPC, 110, 110f Human Microbiome Project, 464–465 Human papillomavirus (HPV), 302, 403 of oropharynx and tonsils, 302 particles, 423 Human prion diseases, 636–642 acquired, 640–641 Creutzfeldt-Jakob disease non-PRNP single nucleotide polymorphisms, 636–637 PRNP gene polymorphisms, 636, 639f susceptibility and phenotype, 636–637 diagnosis, 642–644, 643t cerebrospinal fluid analysis, 643–644 electroencephalography (EEG), 643 neuroimaging, 642–643 pathology, 642 epidemiology and history, 636–642 genetic prion diseases, 638–640 implications of, 640 PrP amyloidosis, 639–640, 639f thalamic degeneration, 638–639 pathological findings in, 637f 809

potential future prion zoonoses, 642 sporadic, 636–638 sporadic fatal insomnia, 638 therapeutics, 644 variably protease-sensitive prionopathy, 637–638 zoonotic transmission, 641–642 Human T-cell leukemia virus type 1 (HTLV-1), 11, 111, 305 Human tumor virology action, 300–302 discovery of, causality, 298–300 Epstein-Barr virus, 303 Hepatitis B virus, 304 Hepatitis C virus, 305 human endogenous retroviruses, 306 human immunodeficiency virus, 305 human papillomaviruses, 302 human T-cell leukemia virus type 1, 305 Kaposi sarcoma–associated herpesvirus, 303–304 Merkel cell polyomavirus, 302–303 retroviral oncogenesis in gene therapy trials, 305 viral carcinogens, principles of, 300 Humoral immune response antigens recognized by, 410–411 Humoral responses, 405 Hybridization probe assay, 453–454 Hydration force, 106 Hyperimmune HBV antiserum plus vaccine, 403 Hypermetabolism, 643 Hypersensitive response (HR), 515 Hyphal anastomosis, 570, 578 Hypometabolism, 643 Hypovirulence, virus induction of, 569 Hypovirulent strains, 568 Hypoxia-inducing factor-1α (HIF-1α), 178 I Iatrogenic CJD, 640–641, 641t Icosahedral symmetry, 52–54, 54f Idling, 367–369 IFI16, 232 IFN-stimulated genes (ISGs), 227 810

ISG15, 238 Imiquimod, 385 Immortalization, 28 Immune mechanisms, 405 Immune risk phenotype, 416 Immunity, 9, 338 Immunization against viral diseases, 400–404 disease/infection. prevention of, 400–403 obstacles, to immunization, 415–417 of elderly, 416 of immunocompromised, 417 of infants, 415–416 of pregnant women, 416–417 structure-based vaccines, 417–418, 417f monoclonal Ab isolation, 417–418 structural biology coupled with computational biology, 418 vaccination, 404–415, 418–433 correlates of protection, 404–405 live virus vaccines, 418–421 vaccine-induced cellular immunity, 413–415 vaccine-induced immunity, mechanisms of, 405–413 vaccine formulation and delivery, 433–438 adjuvants, 433–434 co-delivery or multivalent vaccines, 435 combination approaches, 435–438 delivery vehicles, 434–435 high-throughput sequencing technology, impact of advances in, 436–437 maintaining vaccine availability, 437 modernization of vaccine manufacturing, 436 universal or broadly protective vaccines, 436 vaccine uptake, influences on, 437–438 vaccines, sequential combination of, 436 viral vaccine development and future prospects, history of, 398–400 critical events in history, 400 eradication/elimination, 403–404 immunotherapy, 403 inactivated virus vaccination, origins of, 400, 400f, 401t–402t vaccinology, origins of, 399–400, 399f Immunobinding assays, 460 Immunofluorescence, 479 811

Immunogens, synthetic peptides, 426 Immunoglobulin G (IgG), 406 antibodies, 335 Immunopathology, 248 overactive or dysregulated immune responses, 216–217 Immunotherapy, 403 In vitro neutralizing activity, inhibitory antibodies, 412 In vivo bioluminescence imaging, 35, 35f Inactivated virus vaccines, 421–422 features of, 421–422 future considerations for, 422 origins of, 400, 400f, 401t–402t Incidence rate, 333–334 Inclusion bodies, 30 Incubation period, 339 Induced cellular proliferation, 9 Induced mutation, 38 Induction, 595 Infectious period, 339 Infectious proteins, 4 Inflammasome activation by virus infection, 230–231, 231f Inflammatory cytokines, 235 Influenza A viruses, 344 model for uncoating of, 360–361, 361f uncoating, 108 Influenza HA–based protein vaccines, 425 Influenza HA–mediated membrane fusion, 86f Influenza M1 protein helical array, 74f Influenza mRNA transcription, inhibition, 362, 363f Influenza therapy, 389 Influenza virus, 361 budding, 161, 161f Influenza virus neuraminidase inhibitors, 382–385 Inhibitor of apoptosis (IAP), 529, 541–542 Inhibitory antibodies, in vitro neutralizing activity, 412 Innate antiviral cytokines inflammatory cytokines, 235 interferons, 234, 234f interleukin-6, 235 interleukin-15, 236 812

interleukin-18, 235–236 interleukin-1-beta, 235–236 tumor necrosis factor-alpha (TNFR), 236 Innate immune system, 213–214 cell types evasion strategy mechanisms of DNA vs RNA viruses, 242–243 recruited cells, antiviral defense, 240–241 tissue-resident innate immune cells, antiviral defense, 238–240 viral evasion, 241–242 cell-autonomous virus recognition, 228–232 NOD-like receptors, 230–232, 230f RIG-I–like receptors, 228–230, 229f cell-extrinsic virus recognition, 232–234 Toll-like receptors (TLRs), 232–234, 232f cell-intrinsic antiviral defense mechanisms, 236–238 interferon-induced antiviral defense, 236–238 host protection from infectious pathogens, 227 innate antiviral cytokines, 234–236 inflammatory cytokines, 235 interferons, 234, 234f interleukin-6, 235 interleukin-15, 236 interleukin-18, 235–236 interleukin-1-beta, 235–236 tumor necrosis factor-alpha (TNFR), 236 sensing viral infections, 228 viral sensing through pattern recognition receptors, 228 Inositol phosphate (IP), 182 Inositol polyphosphates, 583 Inoviridae, 609–611, 618 Insect vectors, and plant viruses, 517–519 feeding on virus-infected plants, 517–518 metabolic modifications, benefit for vector, 518–519, 519f nonpersistent and semipersistent transmission, by aphids, 517f–518f plant metabolism, vector transmission, 518 Rice dwarf virus in, 518f Insect viruses biology of, 527–529 biopesticide viruses for insect control, 530–531 classification of, 527–529 813

discovery of, 527–529 expression vectors for foreign genes, 529–530 families, 531–554 Ascoviridae, 546–547 Baculoviridae, 531–542 Dicistroviridae, 552–553 Nodaviridae, 548–550 Nudiviruses, 547 Polydnaviridae, 542–546 Tetraviridae, 551–552 gene delivery, 530 gene therapy, 530 gene transduction, 530 impact of, 527–531 virus evolution, 527–529 virus families infecting insects, 528t virus interactions, 527–529 Intact cellular immunity, 413 Integrated platforms, 458 Interferon stimulated genes (ISGs), 195 Interferon-induced antiviral defense, 236–238 late stages, inhibition, 237 multifunctional antiviral effectors, 238 viral entry inhibition, 236–237 viral genome nuclear translocation, inhibition, 237 viral protein or synthesis, inhibition, 237 Interferon-inducible tetratricopeptide (IFIT), 237 Interferons (IFN), 12, 213–214, 234, 234f, 385 type I, 234 induction, 234 signaling, 234–235 type II, 235 type III, 235 Interleukin-6, 235 Interleukin-15, 236 Interleukin-18, 235–236 Interleukin-1-beta, 235–236 Internal ribosomal entry sites (IRES), 504–506, 529, 553–554 International Committee on Taxonomy of Viruses (ICTV), 21–23 International Plant Protection Convention (IPPC), 496–499 814

Isocitrate dehydrogenase, 178 Isotype, role of, 409 Ivanofsky, Dimitri, 5 J JAKSTAT signal transduction pathway, 12 JC polyomavirus (JCPyV) DNA sequences, 306 Jelly-roll -barrel, 54–55 Jenner, Edward, 399 Jennerian approach, 420 K Kaposi sarcoma (KS), 197 Kaposi sarcoma–associated herpesvirus (KSHV), 197, 297–298, 303–304 derived proteins, 194 Kaumoebavirus, 482–484 kex1 proteases, 566–567 kex2 proteases, 566–567 Kitrinoviricota, 23 Koch, Robert, 1–4 Koprowski, Hilary, 436 L Laboratory-developed tests, 457–458 La Crosse virus, cerebral organoids, 29 λ phages, 592–596 genome, 592, 592f lysogenic cycle, 594–596 CII protein in, 594 CIII proteins in, 594 DNA looping by λ repressor, 595f DNA replication genes of, 595 induction, 595 insertion of DNA into bacterial chromosome, 596f lytic cycle, 593–594 DNA replication, 593 head assembly, 593–594 progeny λ particles, 594 virion, 592–593, 593f Lamivudine, 373 Last Universal Cellular Ancestor (LUCA), 318 Latency-associated nuclear antigen (LANA), 297–298 Latent infection, 192 815

Latent period, 339 LCMV infection, 217 Leakiness, 40–41 Lenacapavir, 377, 378f, 379 Lentiviruses, 24, 207–208 Letermovir, 379–380, 381f Lethal synthesis, 355 Leviviridae, 611–612, 614, 617–618 LGP2, 229 Licensed vaccines, on viral diseases, 404, 404t Light microscopy, diagnostic virology, 451 Lineage-specific virus proteins, core processes, 323–324 Linear and circular genomes, 123 Lipid bilayers, fusion of, 78, 78f Lipid-based carriers, 434 Lipidomics, 186 Lipids catabolism, 184 discovery of, 184 fate of, 184 metabolism, 179–184 synthesis, 180, 181f Lipogenesis, 185 Liposomes, 434 Lipoxygenase products, 180–181 Liquification, 539 Lister, Joseph, 1–4 Live adenovirus vectors, 429 Live recombinant virus vectors, 427 Live virus vaccination, 399, 418–421 advantages of, 419 attenuation of live viruses, genetic basis for, 420–421 disadvantages of, 419–420 isolating attenuated strains, history of methods, 418–419 Liver X receptors (LXRs), 185 Loop-mediated isothermal amplification (LAMP), 454–457, 455f–456f Low-density lipoprotein receptor (LDLR), 185 L2 phages, 613 LTR-mediated activation of proto-oncogene transcription, 295 Lymphocyte recirculation of naive T cells, 254 816

Lymphocytic choriomeningitis virus (LCMV), 12, 217, 405–406 Lysogenic cycle λ phages, 594–596 CII protein in, 594 CIII proteins in, 594 DNA looping by λ repressor, 595f DNA replication genes of, 595 induction, 595 insertion of DNA into bacterial chromosome, 596f Lysogenic phage, 8–9 Lytic cycle λ phages, 593–594 DNA replication, 593 head assembly, 593–594 progeny λ particles, 594 P4, 605 M Macrophages, 228, 239–240 Maintenance of frame (MOF), 565 Major histocompatibility complex class I cell surface expression, nontranscriptional inhibition of, 280 synthesis, class I or II, 279–280 Major histocompatibility complex locus genes/products, 255–256 Malate, 178 Mammalian cells, 425 Mammalian prion diseases, 645–646 Mammalian protein misfolding diseases, 626 Maraviroc, 358–360, 359f Maribavir, 380 Marker rescue, 42 Marseilleviruses, 479 Matrix (M) protein, 74 Mayer, Adolf, 4 MDA5 virus recognition pathways, 229, 229f Measles morbillivirus, 23 Measles virus, 343 cerebral organoids, 29 eradication, requirements for, 348 Mechanical devices, 435 Mechanistic CoP (mCoP), 404–405 817

Mechanistic models, 343 Mediator, 575 Medusavirus, 489–490 genome structure and organization, 489 stages of replication, 489–490, 490f virion structure, 489 Melon necrotic spot virus (MNSV), 509–510 Melting, 539 Melting point analysis, 453 Membrane contact sites (MCS), 507 Membrane extrusion, viral and cellular proteins, 162–163 Membrane immunoassay, antigen detection, diagnostic virology, 452, 452f Membrane proximal external region (MPER), 81, 409 Merkel cell polyomavirus, 302–303 Messenger RNA (mRNA), 7–8 Metabolism diversity in, 173–174 in host fight against virus infection, 186 mevalonate and cholesterol, 182–184, 183f Metabolomics, 186 Metagenomics, 24, 463–465 analyses of viral communities, 619 Mevalonate, 182–184, 183f MF59, 433–434 Microarray technology, 457 Microdomain, 97–99 Microplitis demolitor bracovirus (MdBV), 545 Microtubule-organizing center (MTOC), 107–108 Microviridae, 609, 609f, 613 MIMIVIRE, 477 Mimiviridae, 473–477 complex virions of, 473–474, 473f genome structure and organization, 474–476 stages of replication, 479–480 virion structure, 474, 475f Mitochondrial proton gradient, 177 Mitochondrion, 179 Mitogenic signaling pathways, 296 Mitoviruses, 570 Mobile genetic elements (MGEs), 314–315 818

Modified vaccinia virus Ankara (MVA), 418, 432 Molecular mimicry, 217 Mollivirus, 487–489 genome structure and organization, 487–488 stages of replication, 488–489 virion structure, 487, 488f Molnupiravir, 366f–367f, 369–370 Monkeypox, 389–390 Monoclonal Ab isolation, 417–418 Monocytes, 241 Monodnaviria, 23 Mononegavirales, 504 Mononegaviruses, 570 Monothetic system, 22 Morbillivirus, 348 Mosquito-borne arboviruses, 206 Mouse mammary tumor virus (MMTV), int-1, 295 Movement proteins (MPs), 500 of plant viruses, 324–325 M13 phage, 609–611 adsorption, 610 applications, 611 DNA replication, 610 secretion of, 610f virion, 610 mRNA vaccines, 427 Mu bacteriophages, 605–606 Mucosal antibodies, 412–413 Mucosal virus infections, 412 Multimeric-001 (M-001), 426 Multiple host species, viruses, 341–342, 341t Multiplex assays, nucleic acid amplification assays, 458 Multiplicity of infection (MOI), 33, 33t Multivalent vaccines, 435 Mumps virus, 419 Mutant genotypes, 38 Mutant phenotypes, 38–40 virus genetics drug resistance and dependence, 39–40 host-range, 39 819

neutralization escape, 40 nonsense mutants, 39 plaque morphology, 40 temperature sensitivity, 39 M184V mutation, 373 Mycobacterium tuberculosis, 1–4, 620 Mycoviruses chromosomal genes affecting, 571 negative strand RNA, 570 Myxomatosis virus, 207 N Naive lymphocytes to secondary lymphoid organs, 254–255 Nanoparticles, 434–435 Nanotechnology, P22 bacteriophages, 604 Narnaviridae 20S RNA, 23S RNA, 570 Nascent particles, mechanisms, 163–164 Nasopharyngeal carcinoma (NPC), 303 Natural infection models, 201–202 Natural intrinsic immune defense mechanisms, 227 Natural killer (NK) cells, 213, 240–241, 242f and T-cell recognition, 215 N15 bacteriophages, 606 Nelmes, Sarah, 399 N-ethyl-N-nitrosourea (ENU) mutagenesis, 201 Neurofilament light chain, 644 Neutralization activity, 408–409 Newcastle disease virus (NDV), 424 Next-generation sequencing (NGS), 463–464 Nirmatrelvir, 364f, 389 NLRP3-ASC inflammasome, 231 Nodaviridae, 548–550 Nodaviruses classification and host range of, 548 genome organization, 549 replication, 550 RNA replication spherules and complexes, 551f RNA synthesis, 550 suppression of host RNA silencing, 550 and tetravirus structures, 549f virion structure, 548–549 820

NOD-like receptors (NLRs), 230–232, 230f Non-cell-autonomous pathway (NCAPP), 509 Nonenveloped virus-like particles, 424 Nonmechanistic CoP (nCoP), 404–405 Nonnucleoside inhibitors of polymerases, 375 anti-HIV nonnucleoside reverse transcriptase inhibitors, 375, 376f foscarnet, 375, 376f Nonnucleoside reverse transcriptase inhibitors (NNRTIs), 354, 375 Non-poly(A) mRNAs, 566 Non-PRNP single nucleotide polymorphisms, 636–637 Nonproteolytic maturation events, 162 Nonsense-mediated decay (NMD), 500, 514 Nonstructural protein 1 (NS1), 412 Nonstructural protein 2 (NS2), 362 Nontuberculous Mycobacterium (NTM) infections, 620 Norovirus, 424 NS5A inhibitors, 377 Nuclear egress, 380 Nuclear import, 109–110, 109f Nuclear import and export pathways, 142, 143f Nuclear localization signals (NLSs), 109, 141–142 Nuclear pore complex (NPCs), 108–110, 141 Nuclear retroviral oncoproteins, 294 Nucleic acid amplification assays, 453–458 commercial nucleic acid amplification platforms and tests, 457–458 commercial assays and platforms, 458 government regulation, 457–458 integrated platforms, 458 multiplex assays, 458 contamination, 457 microarray technology, 457 nucleotide sequencing, 457 target amplification, 453–457 polymerase chain reaction, 453–454 RNA amplification assays, 454–457, 454f Nucleic acid genome, 159–161 DNA viruses, 159–160 RNA viruses, 160–161 Nucleic acid vaccines, 426–427 Nucleocapsid, 54 821

Nucleocytoplasmic large DNA virus (NCLDV), 468 Nucleocytoviricota, 467–468, 481–482 Nucleocytoviruses, 469 Nucleopolyhedrovirus OV and BV particles, 532f Nucleoside analogs targeting polymerases, 365–370 acyclovir, 366f–367f, 367 ganciclovir, 366f–367f, 367–369 molnupiravir, 366f–367f, 369–370 Nucleoside-associated mutations (NAMs), 371–373 Nucleotide sequencing, 457 Nucleotide-binding oligomerization domain (NOD), 579 Nudiviridae, 529 Nudiviruses, 547 classification, structure, and genomics of, 547 latency and transmission, 547 O ø29 bacteriophages, 606–607, 607f Obesity, 212 Occluded virus (OV), 531–532, 532f, 538–539 Oligoadenylate synthetases, 238 Oncogene addiction, 301 Oncogenesis, 170 Oncology, 11 “One mRNA one protein” rule, 10 One-step viral growth experiments, 34, 34f Open-source software, 343–344 Opuntia umbralike virus (OULV), 500 Oral polio vaccine type 2 (OPV2), 403–404 Organ culture, 26 Organoid cultures, 26, 29, 29f Orpheovirus, 484–487 genome structure and organization, 486 stages of replication, 486–487 virion structure, 484–486, 485f Ortervirales, 24 Orthoretrovirinae, 24 Orthornavirae, 23 Oryctes virus, 547 Oseltamivir, 384, 389 Oxidative phosphorylation, 177 822

2-Oxoglutarate, 178 2-Oxoglutarate-dependent dioxygenases (2-OGDDs), 178 P p53, 11 Packasome, 599 Pacmanvirus, 482–484 Palm domain, virus replication machinery, 316 Pandoravirus massiliensis, 481 Pandoravirus salinus, 480 Pandoraviruses, 480–482 genome structure and organization, 481–482 stages of replication, 480f, 482 virion structure, 480–481, 480f Papillomaviridae, 125f–128f Papillomavirus pathogenesis, 196, 196f Parallel in-register intermolecular â-sheet/stack (PIRIBS), 628, 629f Paramecium bursaria Chlorella virus 1 (PBCV-1) replication cycle, 470, 471f Paramyxoviridae, 23 Paramyxovirus fusion protein, 82f Pararnavirae, 24 Paritaprevir, 364f Partitiviridae, 567 Parvoviridae, 126f Parvovirus B19, 424 Passage, 27 Passive surveillance, 334 Pasteur, Louis, 1–4 rabies, 4 Pathogen discovery methods, 463, 464f Pathogen-associated molecular patterns (PAMPs), 120–121, 406, 515, 516f Pathogenicity, 339 Pattern recognition receptors (PRRs), 227 viral sensing, 228 P1 bacteriophages, 606 P2 bacteriophages, 604–605 P4 bacteriophages, 604–605 P22 bacteriophages, 603–604 antirepressor, 604 applications, 604 generalized transduction, 603–604 823

genetic diversity and mosaicism, 604 virions, 604 P-bodies, 574 PD pore units (PPUs), 511 Penciclovir, 366f–367f, 370 Penton protein, 477 Peptide transport, 280 Peptidoglycan-cleaving enzymes, 325 Peramivir, 384–385 Perinatal transmission, 341 3-PG (3-phosphoglycerate), 173 PG9, 409–410 PG16, 409–410 Phage lysozyme, 8 Phage-display technology, 611 Phenotypic assays, 461–462, 462t Phenotypic mixing, 46 Phosphatidylcholines (PCs), 182 Phosphatidylethanolamine N-methyltransferase (PEMT), 182 Phosphatidylinositol phosphate (PIP), 182 Phosphatidylinositols (PIs), 182 Phosphatidylserine (PS) lipids, 182 Phosphoenolpyruvate (PEP), 175 Phosphoenolpyruvate carboxykinase (PEPCK), 171, 186 Phosphofructokinase (PFK), 175 Phycodnaviridae, 467–468 Phycodnavirus virion, 473 Phylodynamics, 343–344 Phylogenetic analysis, 344 Physical assays direct particle count, 32 hemagglutination, 32–33 Picornavirus molecular architecture, 54–55 Picornavirus vectors, 429–430 Pithovirus, 484–487 genome structure and organization, 486 stages of replication, 486–487 virion structure, 484–486, 485f PKR-like ER kinase (PERK), 185 Plant viral nanoparticles (VNPs), 520 824

Plant viruses, 494, 496f, 500–504 benefits of and biotechnology, 520–521 delivery vehicles for siRNAs and proteins, 519 for hosts, 519 ecology of, 495–496 epidemics, 496–499, 497t–498t epilogue, 521 Geminivirus, 502 infection cycle and interactions with host cell, 503f genome expression strategies, 500 host responses in defense and immunity, 512–517 antiviral RNAi, 512–513 autophagy, 514 effector-triggered immunity (ETI), 515 pathogen-associated molecular pattern triggered immunity (PTI), 515–516, 516f RNA silencing and associated pathways, 512 secondary sRNA production in antiviral RNAi, 513 SUMOlylation, 514–515 surveillance pathways of cellular mRNA metabolism, 514 ubiquitin proteasome system (UPS), 514–515 unfolded protein response (UPR), 516–517 VSRs with host pathways, 513 and insect vectors, 517–519 feeding on virus-infected plants, 517–518 metabolic modifications, benefit for vector, 518–519, 519f nonpersistent and semipersistent transmission, of viruses by aphids, 517f–518f plant metabolism, vector transmission, 518 Rice dwarf virus (RDV) in, 518f movement of, 509–512 from cellular to whole plant levels, 510f linkage between virus replication and intracellular movement to PD, 509–511 long-distance movement into and out of phloem, 511–512 negative-strand virus infection cycles, 504 nucleic acids in, 496 pandemic, 497t–498t Potyvirus, 502–504 infection cycle and interaction with host cell, 505f recombination in evolution in Potyviridae, 499–500 825

in Tombusviridae, 499–500 (+)RNA virus replication, 507–509 (+)RNA virus translation, 504–507 cap-independent translation, 504–506, 506f cap-independent translation enhancers, 506–507 internal ribosome entry sites, 504–506 taxonomic scheme of, 495f viroids, 512 Plasma membrane, enveloped viruses, 154–155 Plasmacytoid dendritic cells, 241 Plasmaviridae, 613 Plasmodesmata (PD), 500, 507 linkage between virus replication and intracellular movement to, 509–511 P-loop enzymes, 319–320 PM2 bacteriophage, 612–613 Podospora anserina, 579f Polio vaccine development, 406, 407f Polioviruses, 57f, 419 eradication, requirements for, 348 pathogenesis, 205 transcription and assembly of, 158–159 transmission, 205 Poly lactic co-glycolic acid (PLGA), 434 Polydnaviridae, 529, 542–546 Polydnaviriformidae, 542–546 Polydnaviruses classification and structure of, 542–544 contributions to host’s life cycle, 545 genome organization, 544–545 modulation of larval responses during parasitization, 545–546 morphological features of, 544f proviral DNA of, 545 transmission of, 543f Polymerase chain reaction, 453–454 Polyomaviridae, 125f–128f Polyprotein cleavage, inhibition, 362–365 Polyunsaturated fatty acids, 180–181 Porcine circovirus-1 (PCV-1), 436–437 Portal, in bacteriophages, 591–592 Portal rotation model, 599 826

Postassembly modifications and virus release cell-associated and noncanonical spread of infection, 164–165 nascent particles, mechanisms, 163–164 nonproteolytic maturation events, 162 proteolytic cleavage and virus maturation, 161–162 viral and cellular proteins in membrane extrusion, 162–163 Postexposure prophylaxis, 400 Post-Golgi vesicle pH, influenza virus M2 protein, 159 Posttranslational modifications, 144–145 Potato mop-top virus (PMTV), 511 Potato virus X (PVX), 509–510 Potyviridae, recombination in, 499–500 Potyvirus, 502–504 infection cycle and interaction with host cell, 505f Powderject, 435 Poxviridae, 23, 127f Poxvirus Immune Evasion (PIE) domains, 326 Poxviruses, 380 acquisition of multiple membranes, 157–158 therapy, 389–390 vectors, 429, 432 PRD1 bacteriophage, 607–608, 608f Premarket approval (PMA), 457 Presumption of inclusion, 416–417 Primary cell cultures, 27 Primary explant culture, 26 Primary mRNA synthesis DNA viruses of animals, 119–120, 119f RNA viruses of animals, 119–120, 119f Prime-boost strategies, 436 Prion amyloids structures of, 580f HET-s, 580 yeast, 579, 579f Prion propagation chaperone involvement in, 581–582, 581f Hsp70s in, 581 Hsp90s in, 581–582 inositol polyphosphates and, 583 Prions, 626t 827

abnormal PrPd deposition in prion-infected brain tissue, 627f

animal prion diseases bovine spongiform encephalopathy, 644–645 camel prion disease, 645 chronic wasting disease (CWD), 645–646 mammalian prion diseases, 645–646 scrapie in small ruminants, 645 antiprion systems in Saccharomyces cerevisiae, 582–583, 582f Btn2 and Cur1 cure [URE3] isolates, 582–583 Hsp104 disaggregase, antiprion activity of, 583 inositol polyphosphates and prion propagation, 583 inter- or intraspecies barrier to prion transmission, 582 Lug1 lets [URE3] grow, 583 nonsense-mediated decay components Upf1,2,3, 583 ribosome-associated chaperones, 583 Sis1 blocks [PSI+] lethality, 583 chaperone involvement in prion propagation, 581–582, 581f detection of, 646 disease pathogenesis, 632–636 cellular and molecular, 632–635, 634f–635f implications of GPI-anchoring and glycans of, 636 PrPSc toxicity, mechanisms of, 636

evolution of, 624–626 mammalian protein misfolding diseases, 626 prion paradigm to fungi, 626 replication and strains, 626 fibril structures and transmissibility, 630 genetic criteria for, 576f growth, 632, 633f hamster PrP amyloid fibrils, transmissibility and pathogenicity of, 647f [Het-s], 578–579 human prion diseases (See Human prion diseases) inactivation of, 646 movement of, within and between cells, 634f [PIN+], 578 protein isoforms, 625f PrP prions, 626–630 fibril structures and transmissibility, 630 near-atomic structures of infectious tissue-derived prion fibrils, 628–630, 629f PrPC, 630–632 828

structures, 628, 628f [PSI+], 578, 580–581 of Saccharomyces and Podospora, 576–583 spreading within hosts, 632 structures of yeast prion amyloids, 579, 579f transmissible states of proteins, 624 [URE3], 577–578, 580–581 variants, 579, 579f of yeast and fungi, 561, 577t PRNP gene polymorphisms, 636, 639f Productive infection, 192 cytolytic (cytocidal), 192 noncytolytic infections, 192 Program for Monitoring Emerging Diseases (ProMED), 334 Prohormone proteases, 566–567 Prokaryotic cells, production of viral proteins in, 425–426 Prophage, 8–9, 591–592 Prophylactic vaccines, 306 Proportion infectious, 339 Proportion susceptible, 338–339 Protease-resistant protein (PRP) prions, 625–630 amyloidosis, 639–640, 639f fibril structures and transmissibility, 630 near-atomic structures of infectious tissue-derived prion fibrils, 628–630, 629f PrPC, 630–632

structures, 628, 628f Proteases, 320–321, 321f Protein kinase inhibitor, 380 Protein localization, 147 Protein modification, 174 Protein partitioning within cell nuclear import and export nuclear import and export pathways, 142, 143f nuclear localization signals, 141–142 nuclear pore complex, 141 secretory pathway golgi complex, 147 posttranslational modifications, 144–145 protein localization, 147 translocation, 143–144, 144f, 145f 829

transport through, 145–147 Protein purification, 426 Protein-labeled assays, 479 Protein-specific bioassays, 200 Proteins/peptides, vaccines, 425 Proteolytic cleavage, and virus maturation, 161–162 Proteomic studies, 186 Prototype nonacute oncogenic retroviruses, 294–295, 295f Proviral insertion, 295 PrP-scrapie (PrPSc), 633f

implications of GPI-anchoring and glycans of, 636 toxicity, mechanisms of, 636 Pseudoaltermonas, 612 Pseudomonas aeruginosa infection, 612, 620 Pseudomonas putida, 602–603 Pseudotypes, 46 psi sequences, 69 Pyocins, 614–615 Pyruvate kinase, 175 Pyruvates, 171, 173 Q Quantitative assay of viruses biological assays endpoint method, 32 plaque assay, 31–32, 31f–32f physical assays, 30 Quasispecies, 193 Quencher, 453 R rad6 gene, mutation in, 575 Raltegravir, 377, 378f Rapid ER stress-induced export (RESET) mechanism, 632–633 Reactive oxygen species (ROS), 509 Receptor proteins, 97, 98t–99t Receptor-binding domain (RBD), 76–77 Receptor-binding motifs (RBMs), 76–77 Recombinant DNA technology, 287 Recombinant phenotypic assays, 462 Recombination and reassortment, 41–42 Recombination-dependent replication (RDR), 599 830

Rectoanal mucosal lymphoid–associated tissue (RAMALT), 646 Reed-Frost model, 343 Re-emergent viruses, 345–346 REGN-EB3, 407 Relative risk (RR), 335–336 Remdesivir, 366f–367f, 374–375, 389 Reoviridae, 21–22, 512, 529 Reovirus of Cryphonectria, 567–568 Replication-competent vaccinia, 432–433 Replication-competent vectors, 427–430 bacterial vectors, 430 chimeric live virus reassortant and recombinant vaccines, 428–429 herpesvirus vectors, 429 live adenovirus vectors, 429 picornavirus vectors, 429–430 poxvirus vectors, 429 rhabdovirus vectors, 429 Replication-defective vectors, 430–432 adeno-associated virus vectors, 431 adenovirus vectors, 430–431 alphavirus vectors, 431–432 herpesvirus vectors, 432 poxvirus vectors, 432 Reporting efficiency, 334 Repression of E6/E7 expression in cervical cancer cell lines, 302 Reproductive ratio, 342–343 Resistance, 354–355 Respiratory syncytial virus (RSV) infections, 374, 403, 406–407 Respiratory transmission, 341 Restriction endonuclease digestion, 288–289, 288f Restriction enzyme map, 10–11 Restriction mapping, 287 Retinoid X receptors (RXRs), 185 Retroelements, 571–576 Retrointrons, 571 Retroposons, 571 Retrotransposon, 571 Retroviral capsids, intracytoplasmic transport and assembly, 149–151, 150f Retroviral genomes and oncogenes, 287, 288f Retroviral oncogenesis, 291–292 831

in gene therapy trials, 305 Retroviral oncoproteins, signal transduction pathways, 292–294 Retroviral packaging signals, 69 Retroviridae, 24, 129, 130f Retroviruses, 571–576 Candida albicans plasmid retrotransposon and line elements, 576, 580f retrointrons, 571 retroposons, 571 retrotransposon, 571 Schizosaccharomyces pombe, 575–576 Ty elements (See Ty elements) Reverse genetics, 42–46 deep mutational scanning, 46 DNA viruses and reverse-transcribing viruses, 43–44, 43f–44f mutation design, 45–46 RNA viruses, 44–45, 45f Reverse transcriptase, 314 Reverse transcriptase polymerase chain reaction (RT-PCR), 200 Reversion, 40 Revolutionized biology and molecular biology, 287 tool development, tumor virology, 287–289 Revtraviricetes, 24 Rhabdovirus vectors, 429 Rhesus rotavirus (RRV), 107 vaccines, 428 Ribavirin, 374 Ribosomal frameshifting, 565, 573–574 Riboviria, 23–24, 117 Ribozyviria, 23, 117 Rice dwarf virus (RDV), 518f Rice grassy stunt virus (RGSV), 514 Rice yellow mottle virus (RYMV), 512 Rift Valley Fever virus (RVFV), 83 RIG-I–like receptors, 228–230, 229f Rimantadine, 360–361, 361f Rinderpest virus, eradication, requirements, 348 Ritonavir, 383f RNA amplification assays, 454–457, 454f RNA dependent RNA polymerases (RdRp), 193 832

RNA interference (RNAi), 227, 527–529, 568–569 in Aspergillus nidulans, 568–569 in Cryphonectria parasitica, 568 in Neurospora crassa, 568–569 in Sclerotinia sclerotiorum, 568–569 RNA Recognition Motif (RRM) domain, virus replication machinery, 316 RNA silencing, 500, 512 RNA viral genomes viral replication strategies compartmentalization of replication sites, 136 expression and replication, 131–132, 131f–132f host cell factors, 135–136 mechanisms of, 136–137 regulation of, 132–135 structural and nonstructural proteins, 135 transcription, 136–137 RNA virus oncogene action insertional activation of cellular protooncogenes, 294–295 nuclear retroviral oncoproteins, 294 retroviral oncogenes, 291–292 retroviral oncoproteins, signal transduction pathways, 292–294 RNA viruses, 160–161 RNA-dependent RNA polymerases (RdRps), 121, 314 Rolling circle replication initiation endonuclease, 315, 315f Ross, Ronald, 333 Rotavirus, 62f assembly within ER, 156 entry, 87–88, 88f Rubella virus, 400–403, 420 S Saccharomyces cerevisiae, 560 antiprion systems in, 582–583, 582f Btn2 and Cur1 cure [URE3] isolates, 582–583 of Hsp104 disaggregase, 583 inositol polyphosphates and prion propagation, 583 inter- or intraspecies barrier to prion transmission, 582 Lug1 lets [URE3] grow, 583 nonsense-mediated decay components Upf1,2,3, 583 ribosome-associated chaperones, 583 Sis1 blocks [PSI+] lethality, 583 833

Sse1, 582 Totivirus type species L-A of, 561–567 antiviral systems, 566 cap-snatching by Gag separates strands, 565–566 killer phenomenon of, 562f L-A genome structure, 562–564 L-A virion structure, 561–562, 562f N-acetylation of Gag protein by mak3p, 567 Nuc1 and Ski3 block viral lethality in meiosis, 566 positive-strand synthesis, 564–565 posttranslational modification, 566–567 replication (negative-strand synthesis), 567 ribosomal frameshifting, 565 60S subunits, 565 Ski2 antiviral system, 566 stages in replication cycle, 564, 564f transcription reaction, 564–565 translation, 565–566 viral assembly, 567 Ty elements, replication cycle of, 571–573, 572f Saliva and urine assays, 461 Salk and Sabin poliovirus vaccines, 11–12 Salk inactivated virus vaccine, 12 Salk vaccine, 11–12 Salmonella typhimurium, 9 SARS coronavirus (SARS-CoV) infection, 35, 76–77, 77f, 101–102 SARS-CoV1, 12–13 SARS-CoV-2, 15–16, 344 SARSCoV-2 Mpro inhibitor, 365 SARS-CoV-2 therapy, 389 Satellite, dependent, and defective genomes, 124–125 Satellite of tobacco necrosis virus (STNV), 54 Sclerotinia sclerotiorum, 570 Scrapie in small ruminants, 645 Scrapie-associated fibrils (SAF), 625–626 Secondary cell cultures, 27 Secondary sRNA production in antiviral RNAi, 513 Secretory pathway of cell golgi complex, 147 modification of 834

poliovirus, transcription and assembly of, 158–159 post-golgi vesicle pH, influenza virus M2 protein, 159 posttranslational modifications, 144–145 protein localization, 147 translocation, 143–144, 144f, 145f transport through, 145–147 virus assembly and maturation herpesvirus envelopment in the cytoplasm, 156–157 poxvirus acquisition of multiple membranes, 157–158 rotavirus assembly within ER, 156 Segmented and nonsegmented genomes, 123–124 Selective 2' hydroxyl acetylation analyzed by primer extension (SHAPE), 124 Selective-incorporation model, 161 Semliki Forest virus (SFV), 431–432 Senescence, 27 Sequence-based detection technologies, 463 Sequence-based methodologies, 418 Serial interval, 340 Serine protease, 362 Serological surveys, 335 Serology nucleic acid detection, 458–461, 459t agglutination assays, 460 antibody response, kinetics of, 459 binding assays, 460 cerebrospinal fluid serology, 461 in chronic infections, 459 functional assays, 460 immunobinding assays, 460 in reinfection and reactivation, 459 saliva and urine assays, 461 virus-specific IgM assays, 460–461 Serum alanine aminotransferase (sALT), 215–216 Severe acute respiratory syndrome (SARS), 457 Severe fever with thrombocytopenia syndrome virus (SFTSV), 83 Shell domain (“S-domain”), 55 Shield, 32–33 Short interfering RNA (siRNA) libraries, 200–201 Sialic acid receptor-binding domain, 409–410 Signal recognition particle (SRP), 610 835

Simeprevir, 364f Simian retrovirus (SRV), 437 Simian vacuolating virus 40 (SV40) DNA, 10 Simian-human immunodeficiency virus (SHIV) infection, 410–411 Sindbis virus, 412, 431–432 Single- and double-stranded genomes, 122 Single cell RNA sequencing technologies, 200 Single host species, viruses maintained in, 341 Single jelly roll (SJR) capsid protein, 317 Single-stranded DNA gemini-like mycovirus, 571 Single-stranded RNA viruses (ssRNA) chromosomal genes affecting mycoviruses, 571 Hadakaviruses, 570–571 mitoviruses, 570 Narnaviridae 20S RNA, 23S RNA, 570 negative strand RNA mycoviruses, 570 recognition and packaging, 68f, 69 viruses reducing virulence of chestnut blight fungus, 568–570, 569f genome structure, 568–569 infectious complementary DNA clones and biological control of, 570 RNA interference (RNAi), 568–569 virus induction of hypovirulence, 569 virus replication in intracellular vesicles, 568 Yadokari viruses, 570–571 Site-directed mutagenesis, 287 Ski2 antiviral system, 566 Ski proteins, 560–561 Small interfering RNA (siRNA) mechanism, 618 Smallpox, 11 Sofosbuvir, 366f–367f, 374 Solanaceae domain (SD), 515 Solemoviridae, 499–500 Somatic hypermutation (SHM), 405 Specified risk materials (SRMs), 644 Sphingolipids (SLs), 184 Spontaneous mutation, 37–38 Sporadic Creutzfeldt-Jakob disease (sCJD), 636, 638t, 641 Sporadic Creutzfeldt-Jakob tissue infectivity, 640t Sporadic fatal insomnia, 638 Spumaviruses, 153 836

Staphylococcus aureus Pathogenicity Islands (SaPIs), 605 Sterile-responsive element (SRE), 573 Structure-based vaccines, 417–418, 417f monoclonal Ab isolation, 417–418 structural biology coupled with computational biology, 418 Subacute sclerosing panencephalitis (SSPE), 348 Subcellular localization of proteins, 147 Succinate, 178 SUMOlylation, 514–515 Superfamily 3 helicases, 316 Surface lattices, 55 Surveillance pathways of cellular mRNA metabolism, 514 Susceptibility, 338 SYBR Green, 453 Synthetic biology, 191, 200 Synthetic particles, 434–435 Synthetic peptides, immunogens, 426 Synthetic self-assembling polypeptides, 435 T T and B lymphocytes properties, 249, 249t in secondary lymphoid organs, 259–261, 260f T4 bacteriophage, 596–601 antigen display, 600–601, 602f assembly of tail, 601f–602f capsid, 600 core genome, 597 DNA packaging, 599 DNA replication, 599 gene expression and regulation, 597–599 genome, 596, 597f internal proteins, 600 promoters, 598f virion structure and assembly, 599–600 T7 bacteriophages, 602–603 T cells, effector activities of CD8+ effector mechanisms, 273–274 CD8+ T-cell exhaustion/dysfunction, 275 CD4+ TE effector mechanisms, 274–275 regulatory T-cell function, antiviral adaptive immune responses, 275–276 837

T lymphocyte development and selection in thymus, 251, 251f Tape measure protein, 593 Taq polymerase, 454 Taqman assay, 453 Target amplification, 453–457 T-cell activation, 261 T-cell memory, 278 T-cell receptor coreceptor, 257 costimulatory genes and proteins, 257 T-cell response to viruses antigen processing and presentation to T cells, 266 induction of T-cell responses to antigen, 269 by major histocompatibility complex class I pathway, 266–268, 266t major histocompatibility complex class II pathway, 266t, 268–269 Tecovirimat, 380, 381f, 389–390 Tectiviridae, 607–608 Telaprevir, 362–363, 364f Temperate phages, 591–592 Terminal hosts, 342 Terminase, 379 Tetrahedral symmetry, 52–54, 54f Tetraviridae, 551–552 Tetraviruses classification of, 551–552 genome organization, 552, 553f replication, transmission, and pathology, 552 virion structure, 552 T-even phages, circular genetic map, 8 Thalamic degeneration, 638–639 Thioflavin T (ThT), 643–644 3D-correlative fluorescence light and electron microscopy (3D-CLEM), 110 Three-dimensional image reconstruction methods, 50–51 Tissue-resident lymphocytes, 239, 239f Tissue-specific isoforms, induction, 171 Titer, 31 TLR3, 233 TLR7, 233 TLR8, 233 TLR9, 233 838

Tobacco mosaic virus (TMV), 4–6, 4f, 52, 509 Toll-interleukin-1-receptor (TIR) homology domain, 515 Toll-like receptors (TLRs), 228, 232–234, 232f, 406 TLR3, 233 TLR7, 233 TLR8, 233 TLR9, 233 Tomato brown rugose fruit tobamovirus (ToBRFV), 496–499 Tomato bushy stunt virus (TBSV), 52, 55, 507–509, 508f Totivirus type species L-A of S. cerevisiae, 561–567 antiviral systems, 566 cap-snatching by Gag separates strands, 565–566 killer phenomenon of, 562f L-A genome structure, 562–564 L-A virion structure, 561–562, 562f N-acetylation of Gag protein by mak3p, 567 Nuc1 and Ski3 block viral lethality in meiosis, 566 positive-strand synthesis, 564–565 posttranslational modification, 566–567 replication (negative-strand synthesis), 567 ribosomal frameshifting, 565 60S subunits, 565 Ski2 antiviral system, 566 stages in replication cycle, 564, 564f transcription reaction, 564–565 translation, 565–566 viral assembly, 567 Traditional thin-sectioning methods, 50–51 Transcription-based amplification, 454 Transcriptomic studies, 186 Transgenic mice, viral oncogenes, 287 Translocation, 143–144, 144f, 145f Transmissible amyloidosis of Sup35p, 578 of Ure2p, 577–578 Transmissible spongiform encephalopathies (TSEs), 576–577 Transmission ratio, 342–343 Transposable element D (TED), 542 Transpovirons, 477 Triacylglycerols (TAGs), 182 839

Triangulation number (T-number), 52–55, 55f Tricarboxylic acid cycle (TCA), 176–178, 177f Acetyl-CoA, 176–178 citrate, 176–178 intermediates, functions of, 178–179 reducing equivalents and oxidative phosphorylation, 177 Triple gene block (TGB), 509–510 Trofile, 462 Trojan horse strategy, 96 Tumor necrosis factor-alpha (TNFα), 236 Tumor suppressor pathways, inactivation, 296–297 Tumor virology cancer and cell transformation, 286–287 cellular targets of, 289–291, 289f DNA virus oncogene action, 295–298 history of, 287–289 in human, 298–302 and HIV, 302–306 RNA virus oncogene action, 291–295 tool development, revolutionized biology, 287–289 and viral oncogenes, discovery, 287 Tumorigenicity, 28 Turnip mosaic virus (TuMV), 496 Turnip yellow mosaic virus (TYMV), 52, 514 Ty elements, 560–561 expression of, 573–575 +1 ribosomal frameshifting, 573–574 chromosomal genes regulating Ty transcription, 573 control of transcription, 573 host limitations on Ty transposition efficiency, 575 insertion on cellular genes, 573 packaging and assembly, 574 phosphorylation, 574 proteolytic processing, 574 self-imposed copy number control, 574–575 genome structure and expression of, 573f replication cycle of Saccharomyces cerevisiae, 571–573, 572f integration, 572–573 reverse transcription, 572 stucture of, 571 840

Ty5 integration, 575 Type VI secretion systems (T6SS), 615–617, 616f U Ubiquitin proteasome system (UPS), 514–515 Umbravirus like RNAs, 499–500 recombination in evolution of, 501f Umbraviruses, 499–500 Uncoating genome, 108 program, 108 Unfolded protein response (UPR), 516–517 Universal protective vaccines, 436 URE3, 577–578, 580–581 genetic evidence, 577 Ure2p domains of, 577–578 infectious amyloid of, 578 Ure2p domains of, 577–578 infectious amyloid of, 578 recombinant, amyloid filaments of, 578f transmissible amyloidosis of, 577–578 Uronema gigas, 467 V Vaccines sequential combination of, 436 Vaccination, 398, 424–426 enveloped viruslike particles, 424, 424f eukaryotic cells, production of viral proteins in, 425 by gene delivery, 426–433 DNA vaccines, 427 gene-based vectors, limitations of, 432 mRNA vaccines, 427 nucleic acid vaccines, 426–427 replication-competent vectors, 427–430 replication-defective vectors, 430–432 viral vectors as vaccines, 432–433 immunogens, synthetic peptides as, 426 inactivated virus vaccines, 421–422 features of, 421–422 841

future considerations for, 422 live virus vaccines, 418–421 advantages of, 419 attenuation of live viruses, genetic basis for, 420–421 disadvantages of, 419–420 isolating attenuated strains, history of methods, 418–419 nonenveloped virus-like particles, 424 prokaryotic cells, production of viral proteins in, 425–426 with proteins/peptides, 425 protein purification, 426 sequential combination of, 436 types of, 418–433 virus-like particle vaccines, 422–423 advantages of, 423 disadvantages of, 423–424 future considerations for, 423–424 Vaccine hesitancy, 437–438 Vaccine manufacturing, modernization of, 436 Vaccine-induced cellular immunity, 413–415 CD4 T Cells, 414–415 CD8 T cells, 413–414 Vaccine-induced humoral immunity, 406–407, 407f Vaccine-induced immunity mechanisms of, 405–413 affinity, structural and biochemical features of antibodies, 408–409 antibodies active against virus-infected cells, 411 antibodies, adverse effects of, 413 antibodies in vivo, antiviral activity of, 412 antibody escape mechanisms, 411 antibody-mediated virus neutralization, 407–408 CDR3 loops, of antibody variable region, 409 classical in vitro neutralizing activity, inhibitory antibodies, 412 efficient antibody interactions, structural features of antigens critical, 409–410 host antigens, inadvertent induction of antibodies to, 411–412 humoral immune response, antigens recognized by, 410–411 isotype, role of, 409 mucosal antibodies, 412–413 vaccine-induced humoral immunity, 406–407, 407f Vaccinia virus (VACV) protein E3, 23, 326 Vaccinology, origins of, 399–400, 399f 842

Variably protease-sensitive prionopathy (VPSPr), 637–638 Variant CJD (vCJD), 640–642 Varidnaviria, 23, 117 Vector-borne viral infections, 341 Velociraptor, 602 Venezuelan equine encephalitis virus (VEEV), 205–206, 431–432 Vermamoeba vermiformis, 484 Vesicle shuttle mechanism, 105–106, 106f Vesicular stomatitis virus (VSV) mouse model, 408–409 RNP and M, organization of, 70–71, 71f Vestigial esterase domain, 407–408 Vibrio cholerae, 611 Viperin (gene RSAD2), 238 Viral adaptation, 207 Viral antigen recognition B cells antigenicity and immunogenicity, 262–263 B-cell activation by viral antigens, 264–265 B-cell epitopes, 262 primary B-cell repertoire generation, 263–264 by T cells antigen processing and presentation to T cells, 266 induction of T-cell responses to antigen, 269 by major histocompatibility complex class I pathway, 266–268, 266t major histocompatibility complex class II pathway, 266t, 268–269 Viral assembly/egress inhibition of, 379–385 HIV protease inhibitors, 382 influenza virus neuraminidase inhibitors, 382–385 letermovir, 379–380 maribavir, 380 tecovirimat, 380, 381f Viral carcinogens, principles of, 300 Viral clearance and chronic viral infection, 215 tissue damage and disease, 215–216, 216f Viral dynamics, modeling, 343 Viral emergence, 344, 344f Viral entry 843

inhibition of, 356–360 bulevirtide, 356–357 enfuvirtide, 360 fostemsavir, 357–358, 359f maraviroc, 358–360, 359f Viral evasion of cellular (T-cell) immunity cytokine functions, interference cytokine and cytokine receptor homologs, 281 receptor binding and modulation of immune cell function, 281 subverting antigen processing and presentation antigenic peptide generation, 280 class I or II major histocompatibility complex synthesis, 279–280 nontranscriptional inhibition of major histocompatibility complex class I cell surface expression, 280 peptide transport, 280 Viral gene expression inhibition of, 361–365 baloxavir, 362, 363f HCV NS3/4A protease inhibitors, 362–365 Viral genome fossils, 17 Viral genome replication inhibition of, 365, 366f–367f prospects for new inhibitors and targets, 379 Viral genomes structures and organization of DNA vs RNA genomes, 122 linear and circular genomes, 123 positive, negative, and ambisense genomes, 122–123 segmented and nonsegmented genomes, 123–124 single- and double-stranded genomes, 122 Viral glycoproteins in polarized epithelial cells, enveloped viruses, 156 Viral hepatitis therapy, 388–389 HBV therapy, 388 HCV therapy, 388–389 influenza therapy, 389 Viral infections epidemiology of, 13–15, 14t spatial patterns of, 338 Viral metagenomics, 24 Viral molecular clone technologies, 191, 199–200 Viral pathogenesis 844

abortive infection, 192 acute vs chronic infection, 192–193, 193f host response, 193–194 age dependence of virus resistance, 209–210, 210f biological sex, 210–211, 211f clinical observations, 204–205 coinfection, 212 comorbidities on susceptibility, 212 description, 191 disease, 194 foundational principals, 191 genetic determinants, 207–208 integrated effects, viral and host properties, 208–212 invasiveness, 195 latent infection, 192 model systems for human disease, 191 permissive, 192 productive infection, 192 proviral and antiviral host factors, 195 quasispecies, 193 in research areas, 218 sequential stages in infection, 205–207, 205f study of animal models, 201–204 cell culture systems, 199 clinical research, 199 epidemiology, 197–199 functional genomics technologies, 200–201 viral molecular clone technologies, 199–200 susceptible, 192 treatments and vaccines, 191 tropism, 196 variation in host microbiome, 211–212 viral immune evasion, 195–196 viral interactions, host immune system, 212–217, 213f virulence, 194–195 Viral replication complexes (VRCs), 507 antiviral drugs block various stages of, 356, 357f–358f Viral replication cycle, 191 Viral replication strategies 845

cis-acting RNA signals and specificity, 124–125 DNA viral genomes expression and replication, 125–128, 125f–128f latent and persistent infections, 130 mechanisms of, 129–130 regulation of, 128–129 transcription, 129–130 viral oncogenes and neoplastic transformation, 130–131 genome diversity, 117, 118t host cell components, 121–122 host response, 120–121 levels of segmentation, 121 RNA replication, 121 RNA viral genomes compartmentalization of replication sites, 136 expression and replication, 131–132, 131f–132f host cell factors, 135–136 mechanisms of, 136–137 regulation of, 132–135 structural and nonstructural proteins, 135 transcription, 136–137 subcellular sites, 120 unique biology of, 118–120 viral genomes DNA vs RNA genomes, 122 linear and circular genomes, 123 positive, negative, and ambisense genomes, 122–123 segmented and nonsegmented genomes, 123–124 single- and double-stranded genomes, 122 Viral sensing, pattern recognition receptors, 228 Viral vaccines, licensed in United States, 401t–402t Viral virulence, 344–345 Virgiaviridae, 511 Virion-associated nucleic acids (VANA), 496 Virions, 50, 54–55 Virology birth of of immunology, 12 pathogen discovery, 1886–1903, 4–5 plant viruses and chemical period, 5–6, 6f 846

future aspects genomics and the predictive power of sequence analysis, 16–17 pathogen discovery, 17–18 systems biology, 16 virome, 17 history, 1, 2t–3t host-virus interactions and viral pathogenesis, 15 modern concept, development, 7–9 Viromes, 619 Virosomes, 434 Virulence, 194–195 determinants, 196–197 and genes, noncoding promoters or elements, 197 of field isolates, myxoma virus, 1951-1981, 207, 208t phages, 591–592 Virus assembly and maturation intracellular targeting and assembly complex interactions with secretory pathway, 156–158 of enveloped viruses, cellular membranes, 151–156 of enveloped viruses in nucleus, 148–149 intracytoplasmic transport and assembly of retroviral capsids, 149–151 modification of secretory pathway, 158–159 of nonenveloped viruses, nucleus, 147–148 nucleic acid genome, 159–161 post-assembly modifications and virus release, 161–165 cell-associated and noncanonical spread of infection, 164–165 nascent particles, mechanisms, 163–164 nonproteolytic maturation events, 162 proteolytic cleavage and virus maturation, 161–162 viral and cellular proteins in membrane extrusion, 162–163 protein partitioning within cell, 140–147, 141f Virus capsid proteins, 317 double jelly roll capsid proteins, 317 HK97, 317 single jelly roll (SJR) capsid protein, 317 Virus cultivation and assay, 30 hosts for virus cultivation, 26–29 cell culture, 26–29 laboratory animals and embryonated chicken eggs, 26 initial detection and isolation, 25–26 847

modern viral assays and approaches, 34–36 one-step growth experiment, 34 quantitative assay of viruses, 30–33 quantitative considerations in virus assay, cultivation, and experimentation, 33–34, 33t comparison of, 33 dose-response in plaque and focus assays, 33 multiplicity of infection, 33, 33t recognition of viral growth in culture, 29–30, 30f virus cultivation, 30 Virus discovery, 463–465 Virus ecology, 17 Virus entry, structure biology of membrane fusion, 79f, 80f, 81f, 82f, 84f, 85–86, 85f, 86f penetration by nonenveloped viruses, 86–88, 87f, 88f receptors and coreceptors bilayer fusion, 78, 78f class I fusion proteins, 78–83, 79f, 80f, 81f, 82f class II fusion proteins, 83, 84f class III fusion proteins, 83–85, 85f HIV-1 gp120 interaction, 75–76, 77f neuraminidase, 85 viral fusion proteins, 78 Virus genetics defective interfering particles, 46 fundamental genetic concepts, 36–37 essential and nonessential, 37 gain-of-function mutations, 37 genotype and phenotype, 36–37 loss-of-function mutations, 37 selection and screen, 37 wild type, mutations, and mutants, 36 phenotypic mixing and pseudotypes, 46 reverse genetics, 42–46 deep mutational scanning, 46 DNA viruses and reverse-transcribing viruses, 43–44, 43f–44f mutation design, 45–46 RNA viruses, 44–45, 45f study of mutations and mutants, 37–42 complementation, 41 double mutants and siblings, 38 848

induced mutation, 38 leakiness, 40–41 marker rescue, 42 mutant genotypes, 38 mutant phenotypes, 38–40 recombination and reassortment, 41–42 reversion, 40 spontaneous mutation, 37–38 Virus genome evolution, 312 Virus genome replication, 312 Virus hallmark proteins (VHPs), 312 family B DNA polymerases, 314–315 helicases and genome packaging NTPases, 316–317 and implications, origin, 318–319 palm/RRM domain, virus replication machinery, 316 reverse transcriptase, 314 RNA-dependent RNA polymerase, 314 rolling circle replication initiation endonuclease, 315, 315f virus capsid proteins, 317 Virus pan-proteome organization, 328–329, 328f Virus protein evolution dark matter of virus proteomes, 326–327 knowable unknowns, 326–327 unknowable unknowns, 326–327 exaptation and recruitment of host proteins, 327–328 gene exchange, hosts, 312 lineage-specific virus proteins, core processes, 323–324 moderately conserved proteins, virus functions, 319–323 capping enzymes, 322–323, 323f DNA-dependent RNA polymerases and transcription factors, 321–322 P-loop enzymes, 319–320 proteases, 320–321 virus hallmark proteins family B DNA polymerases, 314–315 helicases and genome packaging NTPases, 316–317 and implications, origin, 318–319 palm/RRM domain, virus replication machinery, 316 reverse transcriptase, 314 RNA-dependent RNA polymerase, 314 rolling circle replication initiation endonuclease, 315, 315f 849

virus capsid proteins, 317 virus proteins, virus-host interactions, 324–326 Virus receptors, 97 Virus replication, 248 Virus replication organelles (VRO), 507 Virus structure BPV, 51, 51f–53f closed shells, structure of, 54–67, 56f, 57f elongated shells, 64, 65f frameworks and scaffolds, 59–62, 63f, 64f icosahedral surface organization, 55–59, 60f, 61f, 62f mass production and specificity, 62–64 multishell particles, 64–65, 66f quasi-equivalent icosahedral arrangements, 55, 58f, 59f rearrangements in surface lattices, 65–66, 67f two recurring globular-domain structures, 66–67 cryo-EM bovine papillomavirus, 51, 51f–53f limitation, 51 cryo-ET herpes simplex virus type 1, 51, 52f immature and mature HIV-1 particles, 51, 53f genome packaging dsDNA genomes, 69–70 dsRNA genomes, 70 negative-strand RNA genomes, 70–71, 71f, 72f positive-strand RNA genomes, 68–69, 68f signal, 67–68 post-assembly steps, 75 traditional thin-sectioning methods, 50–51 viral membranes, 71–75, 72f, 73f, 74f virus entry, structure biology of membrane fusion, 85–86 penetration by nonenveloped viruses, 86–88 receptors and coreceptors, 75–85 virus symmetry, 52–54 Virus symmetry, 52–54, 54f Virus taxonomy, 313t current taxonomy, 23–24, 24f history and rationale, 22–23 850

informal groupings and alternate classification schemes, 24, 25t nomenclature, 24 Virus VIGS (virus-induced gene silencing) vectors, 494 Virus-encoded suppressors of RNA silencing (VSRs), 513 Viruses dsRNA viruses (See Double-stranded RNA viruses) intracellular mode of transmission, 560–561 retroviruses, 571–576 Candida albicans plasmid retrotransposon and line elements, 576 retrointrons, 571 retroposons, 571 retrotransposon, 571 Schizosaccharomyces pombe, 575–576 Ty elements (See Ty elements) of simple eukaryotes, 561t single-stranded DNA gemini-like mycovirus, 571 ssRNA viruses (See Single-stranded RNA viruses) Virus-induced cellular dysfunction, 194 Virus-induced cytopathic effects, 29–30, 30f Virus-induced disease, 194 Virus-induced gene silencing (VIGS) vectors, 519–520 Virus-induced immunopathology, 194, 217 Virus-like particles (VLPs) vaccines, 153, 422–423 advantages of, 423 disadvantages of, 423–424 future considerations for, 423–424 Virus-receptor interactions, 76f Virus-specific IgM assays, 460–461 Voxilaprevir, 364f VSV glycoprotein, G (VSV-G), 84, 85f, 408–409 VZV therapy, 387 W Wac protein, 600 Warburg effect, 175–176 Waring blender, 8 West Nile transmission cycle, 342 West Nile virus, 23, 410–411 replication, 180 Wheat soilborne mosaic virus (WSBMV), 512 Whole genome sequencing, 287 851

Wiskott-Aldrich syndrome (WASP)-like protein, 531 X Xenophagy, 227 X-linked agammaglobulinemia, 412 X-ray diffraction patterns, 6 Y Yadokari viruses, 570–571 Yeast prions, 577t structures of, 579, 579f Yellow fever vaccine virus (YFV), 428–429 Z Zaire ebolavirus, 23 ZAP (gene ZC3HAV1), 238 Zidovudine (AZT), 366f–367f, 371–373 biochemical mechanism of, 371, 372f Zika virus, 29, 344 fetal birth defects, 197–199 seropositivity, 197–199 “Zinc-knuckle” modules, 69 Zoonotic transmission, 641–642 Zygotic induction, 8–9

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